Eldew APS-307

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 january 2007 - 15 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Phytosteryldecyltetradecyl N-myristoyl-N-methylaminopropionate
- Substance type: Organic UVCB
- Physical state: Pale yellow waxy solid
- Analytical purity: 100%
- Lot No.: 609074
- Stability under test conditions: stable
- Storage condition of test material: Room temperature, in the dark away from heat and moisture.

Method

Target gene:

Histidine encoding gene for Salmonella.
Tryptophan encoding gene for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 1.2,4.9,20,78,313,1250, and 5000 µg/plate.

Main test:
0.15, 0.31, 0.61, 1.2, 2.4, 4.9, 313, 625, 1250, 2500 and 5000 µg/plate (without S9 mix)
313, 625, 1250, 2500 and 5000 µg/plate (with S9 mix)

Vehicle / solvent:

- Solvent(s) used: Acetone

- Justification for choice of solvent:
Based on the information from the sponsor that the test substance was insoluble in water, the solubility test was performed with DMSO, acetone. And the test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation and the test was performed. In consideration of growth inhibition of acetone to test strains, twice the usual concentration was made and 0.05 mL of the test solution was used. And the tests were conducted in the preincubation method.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine-2HCl

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hrs

NUMBER OF REPLICATIONS: 1 for Preliminary test and 3 for main test.

DETERMINATION OF CYTOTOXICITY : Growth inhibiton.

OTHER EXAMINATIONS:
Counting Procedure: The number of revertant colonies was counted visually due to the precipitate of the test substance. But the
revertant colonies of positive controls were counted with a colony counter.

Evaluation criteria:

Evaluation criteria:
In the main tests carried out twice, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results at each concentration were demonstrated with the. mean and the standard deviation.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 4.9 µg/plate only for TA98, TA1537 without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the precipitate on the plates was observed at 1250 µg/plate and more both with and without metabolic activation.
- During the study period, there was no environmental factor that was thought to have affected the reliability of the study, and there was no deviation from the protocol.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The growth inhibition by the test substance was observed at 4.9 µg/plate and more in S.typhimurium TA98, TA1537 without metabolic activation. And precipitate of the test substance on the plates was observed at 1250 µg/plate and more both with and without metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


ADDITIONAL INFORMATION ON CYTOTOXICITY: Not recorded

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results and Discussion:

In the main tests carried out twice, an increase in the number of revertant colonies (more than twice as many as that of the negative control) was not observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD)in background data, indicating that this study was performed correctly (Appendix). From these results, mutagenicity of the test substance was judged negative. The growth inhibition of the test strains by the test substance was observed at the doses marked with " * " in the Tables, and the precipitate on the plates was observed at 1250 µg/plate and more both with and without metabolic activation. In the sterility test on the test solution and the S9 mix, no growth of bacteria was obsenved.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

From the results described above, it is concluded that Phytosteryl.decyltetradecyl N-myristoyl-N-methylaminopropionate is not mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions, with and without S9.

Executive summary:

Mutagenicity potential of Phytosteryl.decyltetradecyl N-myristoyl-N-methylaminopropionate was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in the presence and absence of S9 in an AMES Test.. In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

The substance is considered to be non-mutagenic with and without S9.