2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

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:

08.02.2010 - 20.04.2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate included in the full study report

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

EC Number:

413-850-6

EC Name:

2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

Cas Number:

123968-25-2

Molecular formula:

C37H56O3

IUPAC Name:

2-{1-[2-hydroxy-3,5-bis(2-methylbutan-2-yl)phenyl]ethyl}-4,6-bis(2-methylbutan-2-yl)phenyl prop-2-enoate

Constituent 2

Reference substance name:

ZIKANOX-549

IUPAC Name:

ZIKANOX-549

Details on test material:

Name: ZIKANOX-549
Manufacturer & Supplier: ZIKO Ltd.
Manufactured: 2009-09-05
Date of Receipt: 2009-10-29
Lot No.: 10909001-001
Purity: 99.55 %
Storage Condition: Room Temperature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: base pair substitution type

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: base pair substitution type

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: base pair substitution type

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: frame shift type

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: frame shift type

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

- Treatment of test item: Treatment of test item was assayed using pre-incubation method. 100 μL of test item, 0.5 mL of S9 mix (or sodium phosphate buffer, pH 7.49) and 0.1 mL of bacterial culture were added to each test tube of dry heat sterilization. This mixture was shaken for 20 minutes using shaking incubator (120 rpm), then was dispensed top agar and was poured onto minimal glucose agar plate (one tube per plate). Vehicle control groups were treated with vehicle only and each of positive control groups was treated with strain-specific positive controls. The solution of the highest concentration of test item (0.1 mL) and S9 mix (0.1 mL), without bacterial culture, were also plated to check sterility. After the top agar was solidified, the plates were inverted, and incubated at 37 °C for about 48 hours. Then the revertants were counted.
- Dose range-finding test: Dose range-finding test was performed with the five test strains at concentration levels of 50, 100, 500, 1000 and 5000 μg/plate both in the absence (S9-) and presence (S9+) mix, and then assayed in triplicate per concentration.
- Main test: 5000 μg/plate both in the absence (S9-) and presence (S9+) of S9 mix was selected as the highest cocentration of the main test considering the result of dose range-finding test. The test item of the highest concentration was serially two-fold diluted to make 5 concentration levels (312.5, 625, 1250, 2500 and 5000 μg/plate), and then assayed in triplicate per concentration

Vehicle / solvent:

Vehicle (Negative Cotrol):
Name: Dimethyl sulfoxide (DMSO)
CAS No.: 67-68-5
Supplier: Sigma-Aldrich, Inc.
Lot No.: 108K01867
Purity: 99.5 %
Storage Condition: Room Temperature

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

4 different positive controls used in the test

Positive control substance:

sodium azide

Remarks:

Additional positive controls : 9-aminoacridine (S9-), 2-(2-furyl)-3-(5-nirto-2-furyl)acrylamide (S9-), 2-aminoanthracene (S9+)

Migrated to IUCLID6: S9-

Details on test system and experimental conditions:

* Positive control I (S9-) *
Name: Sodium azide (NaN3)
CAS No.: 26628-22-8
Supplier: Wako Pure Chemical Industries, Ltd.
Lot No.: DWL5550
Purity: 98 %
Storage condition: Room temperature
Application: TA1535 (0.5 μg/plate)
* Positive control II (S9-) *
Name: 9-aminoacridine (9-AA)
CAS No.: 90-45-9
Supplier: Sigma-Aldrich, Inc.
Lot No.: 106F06681
Purity: 98 %
Storage Condition: Room temperature
Application: TA1537 (80 μg/plate)
* Positive control III (S9-) *
Name: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
CAS No.: 3688-53-7
Supplier: Wako Pure Chemical Industries, Ltd.
Lot No.: PAN0050
Purity: 98 %
Storage Condition: Room temperature
Application: TA98 (0.1 μg/plate), TA100 (0.01 μg/plate) and E.coli WP2uvrA (0.01 μg/plate)
* Positive control IV (S9+) *
Name: 2-aminoanthracene (2-AA)
CAS No.: 613-13-8
Supplier: Sigma-Aldrich, Inc.
Lot No.: S34773-358
Purity: 96 %
Storage Condition: Room temperature
Application: TA98 (0.5 μg/plate), TA100 (1.0 μg/plate), TA1535 (2.0 μg/plate), TA1537 (2.0 μg/plate) and E.coli WP2uvrA (10 μg/plate)

Evaluation criteria:

It was judged to be positive if there was a reproducible increase in the number of colonies in a dose-dependant manner, at least in one test strain with or without the metabolic system, clearly exceeding 2 times of that of control.

Results and discussion

Test results

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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:

not valid

Positive controls validity:

valid

Additional information on results:

- Results were expressed as the mean numbers of colonies ± standard deviation form triplicate plates per dose.
- Growth of bacteria in the used preparation of test item and S9 mix, was not observed in the sterility test.
- There were no inhibition of microbial growth by test item in the form of extracted solution in all test strains. According to these results , 5000 μg/plate was determined as the highest concentration of the main test both in the absence (S9-) and presnece (S9+) of S9 mix.
- The main test was performed in the five test strains at concentration levels of 312.5, 625, 1250, 2500 and 5000 μg/plate both in the absence (S9-) and presence (S9+) of S9 mix. In all test strains, there was no increase in the number of revertant colonies compared to the negative control at any concentration of test item either in the presence or in the absence of metabolic activation system.
- Confirmation test was performed with the same method and concentration levels of the main test both in the absence (S9-) and presence (S9+) of S9 mix.
- The positive controls induced a marked increase in the number of revertant colonies compared to the negative control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Refer to the attached full report.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Test item, ZIKANOX-549, was evaluated for its potential to induce reverse mutation in four histidine auxotroph strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one tryptophan auxotroph strain of Escherichia coli (WP2uvrA).
DMSO was selected as vehicle control and the test item (ZIKANOX-549) was precipitated at all concentrations in the absence of S9 mix (S9-) and in the presence od S9 mix (S9+).
Considering the precipitation of test item and the growth inhibition in the dose range-finding test, the five concentrations of test item were selected between 312.5 and 5000 μg/plate (312.5, 625, 1250 and 5000 μg/plate). There was no contamination from the test item and S9 mix used in the main test and the number of viable cell counts of overnight cultures, estimated with optical density, was 1.0 ~ 3.5x10E9 cells/mL as its proper value.
In the main test, there was no significant increase in the number of revertant colonies as anticipated.
Therefore, it is considered that the test item, ZIKANOX-549, showed no evidence of mutagenic potential at the concentration range tested in five tested strains of Salmonella typhimurium, and in Escherichia coli strain WP2uvrA under the condition of this study.

Executive summary:

- The test item, ZIKANOX-549, was evaluated for its potential to induce reverse mutation in the histidine strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one tryptophan auxotroph strain of Escherichia coli (WP2uvrA).

- The test item was dissolved and diluted with DMSO. Based on the dose range-finding test which was performed at concentrations of 50, 100, 500, 1000 and 5000 μg/plate (the highest concentration), the main test was performed where the final concentrations of test item ranged between 312.5, 625, 1250, 2500 μg/plate and 5000 μg/plate with negative and positive control in the absence and presence of metabolic activation system (S9 mix).

- In this study, there was no significant increase in the number of revertant colonies compared to its vehicle control at any dose in any of the strains. In addition, the antibacterial effects such as decrease in the number of colonies were also not observed in any of the strains. The positive controls induced a marked increase in the number of revertant colonies as anticipated.

- Therefore, these results of the bacterial reverse mutation assay showed that, under the conditions of this study, ZIKANOX-549, was not to induce a reverse bacterial mutation at the concentration tested.