4-hydroxybenzophenone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Oct 2018 to 15 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybenzophenone

EC Number:

214-507-1

EC Name:

4-hydroxybenzophenone

Cas Number:

1137-42-4

Molecular formula:

C13H10O2

IUPAC Name:

4-benzoylphenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%, assumed 100% for testing
Test item storage: At room temperature desiccated

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.

The highest concentration of 4-Hydroxy-benzophenone used in the subsequent mutation assays was the level at which the test item inhibited bacterial growth. At least five different
doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

not specified

Details on test system and experimental conditions:

First Experiment: Direct Plate Assay:
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Second Experiment: Pre-Incubation Assay:
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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:

not applicable

Positive controls validity:

valid

Remarks on result:

other: First Experiment: Direct Plate Assay

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that 4-Hydroxy-benzophenone is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of 4-Hydroxy-benzophenone and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The 4-Hydroxy-benzophenone was a white powder.  The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix and at 5000 μg/plate in the absence and presence of S9-mix in tester strain WP2uvrA.  Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 2500 µg/plate in the tester strains TA1535, TA1537 and TA98.  4-Hydroxy-benzophenone did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 2500 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the

pre-incubation assay.  The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.  These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that 4-Hydroxy-benzophenone is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.