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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD 471 (July 1997), EC B.13/14 and OPPTS
870.5100 (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Name: Dowfax 2A1 solution surfactant
Characterisation: 20-9-2016
Lot: YY00G2OM02

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 fraction
Test concentrations with justification for top dose:
159.375, 318.75, 637.5, 1275, 2550 and 5100 μg (in the presence and absence of metabolic activation).
Test material concentrations based on active ingredient and the top dose set based on initial cytotoxicity screen.
Vehicle / solvent:
distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Evaluation criteria:
Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535 and TA1537
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 negative control value.

Strains TA100 and Escherichia coli WP2 uvrA (pKM101)
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 negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness,
reproducibility) was determined to be non-mutagenic.

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:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Remarks:
water
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
without S9: 9-Aminoacridine hydrochloride hydrate with S9: 2-Aminoanthracene
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
with S9: 2-Aminoanthracene w/o S9: 2-Nitrofluorene
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
with S9: 2-Aminoanthracene w/o S9: Sodium azide
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
w/o S9: 4-Nitroquinoline N-oxide with S9: 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
The test material was non-mutagenic under the conditions of this assay
Executive summary:

The potential of DOWFAX™™ 2A1 Solution Surfactant to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100 and a tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method. DOWFAX™ 2A1 Solution Surfactant was tested in the absence and presence of metabolic activation using sterile distilled water (DW) as the solvent. In the Initial Toxicity-mutation Assay, bacterial cultures were exposed to DOWFAX™ 2A1 Solution Surfactant at concentrations of 1.53, 5.1, 15.3, 51, 153, 510, 1530 and 5100 μg/plate (two plates/concentration) based on the active ingredient (a.i.) concentration. Precipitation was not observed up to the concentration of 5100 μg/plate. Partial inhibition of background lawn was observed at tested concentration of 5100 μg/plate in all the tester strains in absence of metabolic activation system. No inhibition of background lawn was observed up to the tested concentration of 5100 μg/plate in all the tester strains in presence of metabolic activation system. No increase in the number of revertant colonies over the background was observed with the test item in any of the tester strains at any of the tested concentrations in the absence or presence of metabolic activation (5% v/v S9 mix) when compared with the concurrent negative control. The recommended top dose indicated in the guidelines for testing in the Confirmatory Mutation Assay is 5000 μg/plate. Dose calculations for the confirmatory assay were re-calculated using the active ingredient concentration (46.1%) from the concurrently conducted GLP characterization once the results became available. In the Confirmatory Mutation Assay, bacterial cultures were exposed to DOWFAX™ 2A1 Solution Surfactant at concentrations of 159.375, 318.75, 637.5, 1275, 2550 and 5100 μg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/v S9 mix). Precipitation was not observed up to the concentration of 5100 μg/plate. Partial inhibition of background lawn was observed at tested concentration of 5100 μg/plate in all the tester strains in absence of metabolic activation system. No inhibition of background lawn was observed up to the tested concentration of 5100 μg/plate in all the tester strains in presence of metabolic activation system. No increase in the number of revertant colonies over the background was observed in any of the tester strains with the test item at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control. All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system. The DOWFAX™ 2A1 Solution Surfactant stock concentrations 1593.75, 3187.5, 6375, 12750, 25500 and 51000 μg/mL were found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of DOWFAX™ 2A1 Solution Surfactant in the dose formulation were found to be 98.0 %, 95.3 %, 92.5 %, 93.1 %, 96.5 %, 97.6% of the nominal concentrations of dose level T1 (1593.75 μg/mL), T2 (3187.5 μg/mL), T3 (6375 μg/mL), T4 (12750 μg/mL), T5 (25500 μg/mL) and T6 (51000 μg/mL), respectively. The concentrations of DOWFAX™ 2A1 Solution Surfactant in the dose formulation after 4 hours were found to be 100 % and 97.5 % of the 0 hour concentrations of dose level T1 (1593.75 μg/mL) and T6 (51000 μg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient. All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, DOWFAX™ 2A1 Solution Surfactant is concluded to be nonmutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).