4-hydroxybenzophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies carried out according to recognised testing guidelines under GLP certification.

OECD 471

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.  

OECD 473

The objective of this study was to evaluate 4-Hydroxy-benzophenone for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of 4-Hydroxy-benzophenone was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

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

In the first cytogenetic assay, 4-Hydroxy-benzophenone was tested up to 540 and 550 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix, respectively.  Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, 4-Hydroxy-benzophenone was tested up to 200 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 125 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix.  Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.  Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations.  In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay

In the first cytogenetic assay in the absence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

In the presence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone induced a statistically significant, dose related increase in the number of cells with chromosome aberrations both when gaps were included and excluded.  The increases in the number of cells with chromosome aberrations were above the historical control data range.  In addition chromosomal exchanges were observed in the intermediate dose groups.  It should be noted that chromosomal exchanges are comparatively rare spontaneous events (historical control data and Scott et al., 1983 (9)) and greater significance should be attached to the observation of exchange figures. Therefore the increases observed were considered biologically relevant.

Second cytogenetic assay

In the second cytogenetic assay in the absence of S9-mix at the 24 h and 48 h exposure times, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

No effects of 4-Hydroxy-benzophenone on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that 4-

Hydroxy-benzophenone does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In conclusion, this test is valid and 4-Hydroxy-benzophenone is clastogenic in human lymphocytes under the experimental conditions described in this report.  The clastogenic activity is confined to incubations in the presence of S9-mix only.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

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

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

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:

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

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

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

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

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

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

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

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.  

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2018 to 29 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%
Test item storage: At room temperature desiccated

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-Mix

S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies).

The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.

Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

A dose-range finding test was completed. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

First Cytogenetic Assay

Without S9-mix:
460, 520 and 540 µg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix:
100, 450 and 550 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Second Cytogenetic Assay

Without S9-mix:
10, 150 and 200 µg/mL culture medium(24 h exposure time, 24 h fixation time).
10, 75 and 125 µg/mL culture medium (48 h exposure time, 48 h fixation time).

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Experimental study dates: 24 October 2018 to 22 February 2019

Preparation of Test Item:
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item formed a clear light yellow solution in dimethyl sulfoxide.
The final concentration of the solvent in the culture medium was 1.0% (v/v). The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded.
Test item concentrations were used within 2.5 hours after preparation.
Any residual volumes were discarded.

Dose-range Finding Test:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. 4-Hydroxy-benzophenone was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.

Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of 4-Hydroxy-benzophenone for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.

The highest tested concentration was the recommended dose level of 0.01 M (1982 µg/mL).

After 3 h exposure to 4-Hydroxy-benzophenone in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of 4-Hydroxy-benzophenone in the lymphocyte cultures was determined using the mitotic index.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

Cytogenetic Assay:
The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. 4-Hydroxy-benzophenone was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments. To be able to select appropriate dose levels for scoring of chromosome aberrations several repeat assays had to be performed.

First cytogenetic assay:
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of 4-Hydroxy-benzophenone for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.

Based on the mitotic index of the dose-range finding test appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. The follow up experiment was performed with the following modifications of experimental conditions.
 
Second cytogenetic assay:
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of 4-Hydroxy-benzophenone for 24 h and 48 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome Preparation:
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of Slides:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay:
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of Slides for Chromosome Aberrations:
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. Fifty two metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations (see Study plan deviation). In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Solvent for Positive Controls
Hanks’ Balanced Salt Solution (HBSS) (Life technologies, Bleiswijk, The Netherlands), without calcium and magnesium.
All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were thawed immediately before use.

Rationale for test conditions:

As recommended in the OECD 473 guideline

Evaluation criteria:

ACCEPTABILITY CRITERIA:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

STATISTICAL ANALYSIS
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

The Fisher’s exact test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 125, 250, 500, 1000, 1500, 1982

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 250 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 125, 250, 500, 1000, 1500, 1982

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 125µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 24 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 125, 250, 500, 1000, 1500, 1982

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 125 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Dose-Range Finding Test; 48 h exposure time, 48 h fixation time. Concentrations in µg/mL: Control, 125, 250, 500, 1000, 1500, 1982

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 125 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

First Cytogenetic Assay, 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 50, 200, 400, 500, 600, 700, 800, MMC-C; 0.5 µg/mL, MMC-C; 0.75 µg/mL

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 50 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

First Cytogenetic Assay, 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 50, 200, 400, 500, 600, 700, 800, MMC-C; 0.5 µg/mL, MMC-C; 0.75 µg/mL

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 50 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Cytogenetic Assay 1A, 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 100, 200, 400, 450, 500, 550, 600, 650, CP; 10 µg/mL

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 100 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Cytogenetic Assay 1A, 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 100, 200, 400, 450, 500, 550, 600, 650, MMC-C; 0.5 µg/mL, MMC-C; 0.75 µg/mL

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 100 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Cytogenetic Assay 1B, 3 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 100, 200, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, MMC-C; 0.5 µg/mL, MMC-C; 0.75 µg/mL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 200 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Second Cytogenetic Assay, 24 h exposure time, 24 h fixation time. Concentrations in µg/mL: Control, 10, 50, 75, 100, 125, 150, 175, 200, MMC-C; 0.2 µg/mL, MMC-C; 0.3 µg/mL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 10 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Remarks:

Second Cytogenetic Assay, 48 h exposure time, 48 h fixation time. Concentrations in µg/mL: Control, 10, 50, 75, 100, 125, 150, 175, 200, MMC-C; 0.1 µg/mL, MMC-C; 0.15 µg/mL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 10 µg/mL onwards

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Dose-range Finding Test

At a concentration of 0.01 M (1982 µg/mL) 4-Hydroxy-benzophenone precipitated in the culture medium.  Therefore, a concentration of 1982 µg/mL was used as the highest concentration of 4-Hydroxy-benzophenone.  The pH and osmolarity of a concentration of 500 µg/mL were 7.773 and 403 mOsm/kg respectively (compared to 7.885 and 419 mOsm/kg in the solvent control).

In the dose-range finding test blood cultures were treated with 125, 250, 500, 1000, 1500 and 1982 µg 4-Hydroxy-benzophenone/mL culture medium with and without S9-mix.

Table 1 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the negative control item.

First Cytogenetic Assay

Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:

Without and with S9-mix: 50, 200, 400, 500, 600, 700 and 800 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Table 2 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the positive or negative control items.

 

In the absence of S9-mix, no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 400 µg/mL not enough cytotoxicity was observed (31%), whereas the next higher concentration of 500 µg/mL was too toxic for scoring (68%).  

In the presence of S9-mix, no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 500 µg/mL not enough cytotoxicity was observed (34%), whereas the next higher concentration of 600 µg/mL was too toxic for scoring (89%).

The experiment was repeated in cytogenetic assay 1A with the following dose levels:

Without and with S9-mix: 100, 200, 400, 450, 500, 550, 600 and 650 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Table 3 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the positive or negative control items.

In the absence of S9-mix, no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 550 µg/mL not enough cytotoxicity was observed (32%), whereas the next higher concentration of 600 µg/mL was too toxic for scoring (93%).

This part of the experiment was repeated in cytogenetic assay 1B with the following dose levels:

Without S9-mix: 100, 200, 400, 420, 440, 460, 480, 500, 520, 540, 560 and 580 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Table 4 (Appendix 1) shows the mitotic index of cultures treated with various test item concentrations or with the positive or negative control items.

The following dose levels were selected for scoring of chromosome aberrations:  

Without S9-mix       :       460, 520 and 540 µg/mL culture medium (3 h exposure time, 24 h fixation time).

With S9-mix       :       100, 450 and 550 µg/mL culture medium (3 h exposure time, 24 h fixation time).

In the absence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix (Appendix 1, Table 5).

In the presence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded (Appendix 1; Table 6).  The increases in the number of cells with chromosome aberrations were above the historical control data range.  In addition chromosomal exchanges were observed in the intermediate dose group.  A dose related trend was observed (p=0.014).

Both in the absence and presence of S9-mix, 4-Hydroxy-benzophenone did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Second Cytogenetic Assay

To obtain more information about the possible clastogenicity of 4-Hydroxy-benzophenone, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to 4-Hydroxy-benzophenone in the absence of S9-mix for 24 or 48 hours.  The following dose levels were selected for the second cytogenetic assay:

Without S9-mix       :       10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).

Table 7 (Appendix 1) shows the mitotic index of cultures treated with various 4-Hydroxy-benzophenone concentrations or with the positive or negative control items.

 

Based on these observations the following doses were selected for scoring of chromosome aberrations:

Without S9-mix       :       10, 150 and 200 µg/mL culture medium (24 h exposure time, 24 h fixation time).

                                       10, 75 and 125 µg/mL culture medium (48 h exposure time, 48 h fixation time).

4-Hydroxy-benzophenone did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Appendix 1; Table 8 - 9).

4-Hydroxy-benzophenone did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

 

Evaluation of the Results

The ability of 4-Hydroxy-benzophenone to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments.  The highest concentration analyzed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater.

The mitotic indices of cultures treated with various 4-Hydroxy-benzophenone concentrations or with the negative control items are presented in Table 1 - 4 and Table 7 (Appendix 1).  The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of 4-Hydroxy-benzophenone are presented in Table 5, Table 6 and Table 8 - 9 (Appendix 1).  Duplicate cultures are indicated by A and B.  The criteria according to which the aberrations were classified are outlined in Appendix 2.  Appendix 3 presents the statistical evaluations of the test results.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Appendix 4).  The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Appendix 6).  The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells (see Appendix 3).  In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay

In the first cytogenetic assay in the absence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

In the presence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone induced a statistically significant, dose related increase in the number of cells with chromosome aberrations both when gaps were included and excluded.  The increases in the number of cells with chromosome aberrations were above the historical control data range.  In addition chromosomal exchanges were observed in the intermediate dose groups.  It should be noted that chromosomal exchanges are comparatively rare spontaneous events (historical control data and Scott et al., 1983 (9)) and greater significance should be attached to the observation of exchange figures. Therefore the increases observed were considered biologically relevant.

Second cytogenetic assay

In the second cytogenetic assay in the absence of S9-mix at the 24 h and 48 h exposure times, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

No effects of 4-Hydroxy-benzophenone on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that 4-Hydroxy-benzophenone does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:

In conclusion, this test is valid and 4-Hydroxy-benzophenone is clastogenic in human lymphocytes under the experimental conditions described in this report. The clastogenic activity is confined to incubations in the presence of S9-mix only.

Executive summary:

The objective of this study was to evaluate 4-Hydroxy-benzophenone for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of 4-Hydroxy-benzophenone was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

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

In the first cytogenetic assay, 4-Hydroxy-benzophenone was tested up to 540 and 550 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix, respectively.  Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, 4-Hydroxy-benzophenone was tested up to 200 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 125 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix.  Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.  Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations.  In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay

In the first cytogenetic assay in the absence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

In the presence of S9-mix at the 3 h exposure time, 4-Hydroxy-benzophenone induced a statistically significant, dose related increase in the number of cells with chromosome aberrations both when gaps were included and excluded.  The increases in the number of cells with chromosome aberrations were above the historical control data range.  In addition chromosomal exchanges were observed in the intermediate dose groups.  It should be noted that chromosomal exchanges are comparatively rare spontaneous events (historical control data and Scott et al., 1983 (9)) and greater significance should be attached to the observation of exchange figures. Therefore the increases observed were considered biologically relevant.

Second cytogenetic assay

In the second cytogenetic assay in the absence of S9-mix at the 24 h and 48 h exposure times, 4-Hydroxy-benzophenone did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

No effects of 4-Hydroxy-benzophenone on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that 4-Hydroxy-benzophenone does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In conclusion, this test is valid and 4-Hydroxy-benzophenone is clastogenic in human lymphocytes under the experimental conditions described in this report.  The clastogenic activity is confined to incubations in the presence of S9-mix only.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available