Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

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:

2010-10-20 to 2010-11-18

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

EC Number:

700-321-2

Molecular formula:

C30H26O6Zn, C46H42O6Zn, C62H58O6Zn

IUPAC Name:

hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

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):

NA

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):

NA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (phenobarbital (PB)- and ß-naphtoflavone (BNF)-induced)

Test concentrations with justification for top dose:

Pre-Experiment for Toxicity
Strains: Salmonella typhimurium TA98 and TA100 strains
Concentrations: 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were tested for toxicity and mutation induction in triplicates in the presence and absence of metabolic activation system (±S9-mix). The test item was dissolved in DMSO.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium TA100 and TA1535; dissolved in distilled water

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine (NPD)

Remarks:

S. typhimurium TA98; dissolved in DMSO

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

S. typhimurium TA1537; dissolved in DMSO

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA; dissolved in distilled water

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 h at 37 +/- 1.5 °C in darkness.

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- growth inhibition

Evaluation criteria:

Evaluation of experimental data
The colony numbers on the control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated.
The Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values were used for this calculation).

Evaluation of Results
The test is considered acceptable if for each strain:
– the bacteria demonstrate their typical responses to crystal violet and ampicillin
– the control plates without S9 mix are within the historical control data range
– corresponding background growth on both negative control and test plates occurs
–the positive controls show a distinct enhancement over the control plate
A test item is considered mutagenic if:
– a dose–related increase in the number of revertants occur and/or
– a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
A biologically relevant increase is described as follows:
– if in strain TA 100 the number of reversions is at least twice as high when compared to the spontaneous reversion rate of the solvent control plates,
– if in strains TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive
response at any of the test points is considered non-mutagenic in this system.

Statistics:

The colony numbers on the control, positive control and the test plates were determined, the mean values and appropriate
standard deviations were calculated.
The Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control
(the exact and not the rounded values were used for this calculation).

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Using the plate incorporation method (Experiment I), none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. Using the pre-incubation method (Experiments II and III), none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. Strong cytotoxic effect (inhibited background lawn development and reduced number of revertant clonies) of the test item was observed in the Experiment II using the pre-incubation method. The observed cytotoxicity was confirmed in the Experiment III. Precipitate was observed in all of the tester strains at 5000 µg/plate concentration with and without metabolic activation in the Experiment I and II. Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colonies of the untreated and solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The test bacteria demonstrated their typical responses to crystal violet and ampicillin. The tests were considered to be valid.

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc is considered non-mutagenic in this bacterial reverse mutation assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Conclusions:

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

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was dissolved in Dimethyl sulfoxide. In the Initial Mutation Test and Confirmatory Mutation Test the tested concentrations were: 5000; 1581; 500; 158.1; 50; 15.81 and 5 µg/plate. In the Complementary Confirmatory Mutation Test the tested concentrations were: 1581; 500; 158.1; 50; 15.81; 5; 1.581 and 0.5 µg/plate.

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc in three independent experiments, in a plate incorporation test (Experiment I, Initial Mutation Test) and in two pre-incubation tests (Experiment II, Confirmatory Mutation Test and Experiments III, Complementary Confirmatory Mutation Test). Experiments I and II were carried out in the presence and absence of a post mitochondrial supernatant prepared from the livers of phenobarbital/β-naphthoflavone-induced rats (±S9-mix), while Experiment III was carried out only in absence of this metabolic activation. The concentrations, including the controls, were tested in triplicates. Using the plate incorporation method (Experiment I), none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. Using the pre-incubation method (Experiments II and III), none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. Strong cytotoxic effect (inhibited background lawn development and reduced number of revertant colonies) of the test item was observed in the Experiment II using the pre-incubation method. The observed cytotoxicity was confirmed in the Experiment III. Precipitate was observed in all of the tester strains at 5000 µg/plate concentration with and without metabolic activation in the Experiment I and II. Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colonies of the untreated and solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The test bacteria demonstrated their typical responses to crystal violet and ampicillin. The tests were considered to be valid.