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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:
From June 16, 2009 to July 08, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
Histidine
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:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
- In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No toxicity or positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.
- In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed at 1500 μg per plate. No appreciable toxicity was observed.
Vehicle / solvent:
Tetrahydrofuran (THF) for the test substance and DMSO for the positives controls
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
- The assay was performed in two phases, using the plate incorporation method. The first phase (1.), the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase (2.), the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.
1. Vehicle control, positive controls and eight dose levels of the test substance were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
2. Six dose levels of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control and positive controls were plated in triplicate.
- The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.
Rationale for test conditions:
Guidelines
Dose-range finding test
Cytotoxicity - Precipitation
Evaluation criteria:
- Scoring:
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
- Evaluation:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhymurium TA98, TA100, TA1535 and TA1537 + E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1. Initial Toxicity-Mutation Assay:
The test substance formed workable suspensions in THF from 60 to 200 mg/mL, a soluble but cloudy solution at 20 mg/mL and soluble and clear solutions from 0.060 to 6.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No background lawn toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

2. Confirmatory Mutagenicity Assay:
The test substance formed workable suspensions in THF from 20 to 200 mg/mL, a soluble but cloudy solution at 6.0 mg/mL and a soluble and clear solution at 2.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No appreciable toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

- All criteria for a valid study were met as described in the protocol.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.
Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance, 'di-C18 -22 AAEMIM-MS', in an Ames test, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 uvr A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No toxicity or positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. The test substance formed workable suspensions in the vehicle (tetrahydrofuran) from 20 to 200 mg/mL, a soluble but cloudy solution at 6.0 mg/mL and a soluble and clear solution at 2.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No appreciable toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The negative and positive controls gave results within the expected range; hence the experiment was considered valid. Under the study conditions, the test substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2009).