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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 471: GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions from Aroclor exposed rats
Test concentrations with justification for top dose:
Test #1 (-S9): 0, 0.05, 0.0158, 0.005, 0.00158, 0.0005, 0.000158 uL/plate; (+S9): 0.5, 0.158, 0.05, 0.0158, 0.005, 0.00158 uL/plate
Test #2 (-S9): 0, 0.05, 0.0158, 0.005, 0.00158, 0.0005, 0.000158 uL/plate; (+S9): 0.5, 0.158, 0.05, 0.0158, 0.005, 0.00158 uL/plate
Test #3 (-S9): 0, 0.005, 0.00158, 0.0005, 0.000158, 0.00005, 0.0000158 uL/plate; (+S9): 0.158, 0.05, 0.0158, 0.005, 0.00158, 0.0005 uL/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene and ICR191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays):
- his+ and trp+ revertants
DETERMINATION OF CYTOTOXICITY
- Method: concentrations eliciting less than 80% viability (as determined by a proportional reduction of ATP in a luminescence assay) and/or impacting the background lawn of bacterial growth
Evaluation criteria:
In order for a test substance to be considered positive in the bacterial gene mutation test, there must be a concentration-related increase over the range tested and/or a reproducible increase of 3-fold or higher over the background, at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. If neither of the above is true, the test article was deemed to be negative.
If the test article was deemed negative in all five strains both in the presence and absence of metabolic activation system, a confirmatory run was performed. If the test article was deemed to be negative in the confirmatory run also (ie there is no reproducible increase of 3 fold or higher over the background, at any concentration, in the number of revertant colonies per plate in any strain with or without metabolic activation system) then the test article was deemed negative for mutagenicity.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitution for frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic.
Statistics:
For cytotoxicity, the mean % viability and standard deviation were calculated. For the main study and confirmatory run the mean revertant colonies, fold change, and standard deviation per treatment group were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at/above 18.5 nL/mL Exxal 13 (-S9), and at/above 585 nL/mL Exxal 13 (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The bacterial reverse mutation test to assess the genotoxicity of Exxal 13 was negative.
Executive summary:

Exxal 13 was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98 and 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver S9 fraction for metabolic activation. Three tests were performed:Test #1 [(-S9): 0, 0.05, 0.0158, 0.005, 0.00158, 0.0005, 0.000158 uL/plate; (+S9): 0.5, 0.158, 0.05, 0.0158, 0.005, 0.00158 uL/plate], Test #2 [(-S9): 0, 0.05, 0.0158, 0.005, 0.00158, 0.0005, 0.000158 uL/plate; (+S9): 0.5, 0.158, 0.05, 0.0158, 0.005, 0.00158 uL/plate], Test #3 [(-S9): 0, 0.005, 0.00158, 0.0005, 0.000158, 0.00005, 0.0000158 uL/plate; (+S9): 0.158, 0.05, 0.0158, 0.005, 0.00158, 0.0005 uL/plate].

Test concentrations at/above 18.5 nL/mL Exxal 13 (-S9), and at/above 585 nL/mL Exxal 13 (+S9) were found to be cytotoxic. The results suggest that Exxal 13 is negative for mutagenicity under the experimental conditions of this study, as it does not induce a 3-fold increase in revertancy compared to negative controls in the presence or absence of S9 in the standard run or the confirmatory test.