Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-2019
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
Light yellow solid (powder)

Method

Target gene:
Salmonella typhimurium (S. typhimurium, ST) and Escherichia coli (E.coli, EC), the bacterial strains were purchased from Molecular Toxicology
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
The Ames test was conducted with Chiguard GA403 at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 μg/plate, with the high level being the standard limit for this test.
Vehicle / solvent:
The test substance was found to be suspendable in dimethyl sulfoxide (DMSO) which was used as the vehicle control.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: ICR 191 Acridine; Daunomycin; 2-Aminoanthracene
Details on test system and experimental conditions:
Bacteria (Test Systems)
Fresh bacterial suspension cultures in nutrient broth were prepared so that they were in the late exponential phase of growth at the time of use (approximately 1 x 109 bacteria/mL). Bacterial growth was evaluated by spectrophotometric optical density measurement.

Main Test
The initial test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of a minimal agar plate:
 100 μL of the prepared test substance solutions, negative (vehicle) control, or prepared positive control substance
 500 μL S9 mix or substitution buffer
 100 μL bacteria suspension (ST or EC)
 2000 μL overlay agar maintained at approximately 45°C
Plates were prepared in triplicate at each experimental point and uniquely identified. After pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approximately 37oC until growth was adequate for enumeration (approximately 65 hours). Appropriate sterility control check plates (treated with critical components in the absence of bacteria) were included as a standard procedural check.
Confirmatory Test
The confirmatory test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at approximately 37°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The study design for the confirmatory test, including strains, dose levels etc. was as described above for the initial (main) test.
Evaluation criteria:
Evaluation of Toxicity
Toxic effects of the test substance are indicated by the partial or complete absence of a background lawn of non-revertant bacteria (colony counts, if any, should not be reported) or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range. Where precipitation obscures observations on the condition of the background lawn, the lawn can be considered normal and intact if the revertant colony counts are within the expected range based on results for lower dose levels and historical control counts for that strain.

Evaluation of Mutagenicity
For each experimental point, the Mutation Factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:
The results were considered positive (i.e., indicative of mutagenic potential) if:
 The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA100, and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
 The above increase must be dose related and/or reproducible, i.e., increases must be obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion or with different methodologies).
If the second criterion is not met, the results may be classified as equivocal, and further testing may be appropriate.
A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies is considered to be non-mutagenic in this test system.

Results and discussion

Test resultsopen allclose all
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
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
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
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
Species / strain:
E. coli WP2 uvr A
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

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid.

Signs of precipitation were observed at doses ≥ 500 μg/plate for TA1537, TA98, TA100 and E. coli, with and without S9 using the plate incorporation and pre-incubation method. In addition, signs of precipitation were observed for strain TA1535 ≥ 1580 μg/plate with and/or without S9 in the plate incorporation method and ≥ 500 μg/plate with and without S9 in the pre-incubation method. Precipitation which obscured assessment of the background lawn was noted for all strains at doses ≥ 1580 μg/plate with the exception of TA1535 at 1580 μg/plate with S9 in the pre-incubation method.

Individual plate contamination, that did not obscure plate counts, was observed for strain TA1535 at 15.8 μg/plate and TA100 at 1.58 μg/plate without S9 in the pre-incubation method. Slight decreases of revertant counts indicating toxicity were observed for strain TA1537 at 5000 μg/plate in the pre-incubation method without S9 and for strain TA98 ≥ 1580 μg/plate with and/or without S9 in the plate incorporation and pre-incubation method.

For all strains, at least five non-toxic dose levels were evaluated; therefore bacterial mutagenicity was adequately assessed.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E.coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method.

Applicant's summary and conclusion

Conclusions:
Based on these findings and on the evaluation system used, Chiguard GA403 did not elicit evidence of bacterial mutagenicity in the Ames assay.