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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 28, 2017 - February 12, 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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Composition: Benzenesulfonic acid, 2,4,5-trichloro-, sodium salt (STB)
CAS #53423-65-7
Purity: 90.5%
Other ingredients - 9.5%
Physical Description: White powder
pH: 7.11
Stability: Test substance was expected to be stable for the duration of testing.
Expiration Date: October 2019

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
chemically-induced rat liver S9 mix
Test concentrations with justification for top dose:
0, 1.58, 5.5, 15.8, 50, 158, 500, 1580, and 5000 μg/plate, with the top dose considered the limit test for this model.
Vehicle / solvent:
Without metabolic activation: Sterile water
With metabolic activation: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strains TA100 and TA1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR 191 Acridine
Remarks:
TA1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
TA98 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
All strains; with metabolic activation
Details on test system and experimental conditions:
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 37°C 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.
Rationale for test conditions:
The background lawn for vehicle control plates should appear normal (i.e., slightly hazy with abundant microscopic non-revertant bacterial colonies). The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012).
Evaluation criteria:
Mutagenic activity of the test item was assessed by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. Toxic effects of the test substance were indicated by the partial or complete absence of a background lawn of non-revertant bacteria, 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.

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.
Statistics:
Product Safety Labs calculated means and standard deviations for all quantitative data collected.

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 nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle 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:
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.

 

No signs of precipitation were noted in any of the strains. Contamination which did not obscure counts was seen for TA1537 at dose 5000 µg/plate with S9 in the pre-incubation method. Contamination did not affect mutagenic evaluation. For all strains, at least eight non-toxic doses were evaluated; hence 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:
The test substance did not elicit evidence of bacterial mutagenicity in the Ames assay.
Executive summary:

In a GLP guideline bacterial reverse mutation assay using the plate incorporation and pre-incubation methods conducted according to OPPTS 870.5100, OECD 471, and EC method B.13/14, the substance did not elicit evidence of bacterial mutagenic activity with or without rat liver S9 metabolic activation with strains TA1535, TA1537, TA98 , TA100 or E. coli WP2 uvrA.