2-benzoylbenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Dec 2018 to 24 Mar 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-benzoylbenzoic acid

EC Number:

201-612-2

EC Name:

2-benzoylbenzoic acid

Cas Number:

85-52-9

Molecular formula:

C14H10O3

IUPAC Name:

2-benzoylbenzoic acid

Specific details on test material used for the study:

Appearance: Off-white powder
Purity/Composition: 98.8%
Test item storage: At room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guideline (OECD).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2018) are presented below:
Dose-range finding study: age 27, AGT = 12.3 h
First cytogenetic assay: age 28, AGT = 13.1 h
Cytogenetic assay 1A: age 25, AGT = 12.8 h
Cytogenetic assay 1B: age 27, AGT = 12.3 h
Second cytogenetic assay: age 28, AGT = 13.1 h

Cell Culture:
Blood samples:
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures:
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.

Environmental conditions:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 43 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 31.9 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First Cytogenetic Assay:

Without S9-mix : 250, 1900 and 2050 µg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix : 250, 1800 and 2000 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Second Cytogenetic Assay:

Without S9-mix: 125, 500 and 650 µg/mL culture medium (24 h exposure time, 24 h fixation time).
125, 800 and 1000 µg/mL culture medium (48 h exposure time, 48 h fixation time).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, also functioning as negative control

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

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. 2-Benzoylbenzoic acid 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 2-Benzoylbenzoic acid 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 (2262 µg/mL).

After 3 h exposure to 2-Benzoylbenzoic acid 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 2-Benzoylbenzoic acid 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 (3 h in the presence of S9-mix and 24 h exposure time) or was determined by the solubility (3 h in the absence of S9-mix and 48 h exposure time).

Cytogenetic Assay:
The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. 2-Benzoylbenzoic acid 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 for the first cytogenetic assay.

First cytogenetic assay
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of 2-Benzoylbenzoic acid 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 and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. 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 2-Benzoylbenzoic acid 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 (3 h in the presence of S9-mix and 24 h exposure time). Also cultures treated with an intermediate dose were examined for chromosome aberrations. In case 2-Benzoylbenzoic acid was not cytotoxic or difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium (3 h in the absence of S9-mix 48 h exposure time).

4.7.6. 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. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. 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.

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.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

2-Benzoylbenzoic acid is clastogenic in human lymphocytes under the experimental conditions described in this report. 2-Benzoylbenzoic acid may have the potential to disturb mitotic processes and cell cycle progression.

Remarks on result:

other:

Remarks:

2-Benzoylbenzoic acid is clastogenic in human lymphocytes under the experimental conditions described in this report. 2-Benzoylbenzoic acid may have the potential to disturb mitotic processes and cell cycle progression.

Any other information on results incl. tables

Dose-range Finding Test

In the dose-range finding study, blood cultures were treated with 125, 250, 500, 1000, 2000 and 2262 µg 2-Benzoylbenzoic acid/mL culture medium with and without S9-mix. At aconcentration of 2262 µg/mL (3 h fixation time) and at a concentration of 1000 µg/mL andupwards (24 h and 48 h fixation time) 2-Benzoylbenzoic acid precipitated in the culturemedium.

The pH and osmolarity of a concentration of 1000 µg/mL were 7.5 and 388 mOsm/kg respectively (compared to 7.9 and 257 mOsm/kg in the solvent control).

Table 1 (Appendix 1) shows the mitotic index of cultures treated with various 2-Benzoylbenzoic acid 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 S9-mix : 250, 1000, 1250, 1500, 1750 and 2000 µg/mL culture medium (3 h exposure time, 24 h fixation time).

With S9-mix : 250, 500, 750, 1000, 1250 and 1500 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 2-Benzoylbenzoic acid 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 1750 µg/mL not enough cytotoxicity was observed (48%), whereas the next higher concentration of 2000 µg/mL was too toxic for scoring (69%).

In the presence of S9-mix no toxicity or precipitate was observed at any of the dose levels tested.

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

Without S9-mix : 250, 1000, 1500, 1600, 1700, 1800 and 1900 µg/mL culture medium (3 h exposure time, 24 h fixation time).

With S9-mix : 250, 750, 1000, 1500, 1700, 2000 and 2262 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 2-Benzoylbenzoic acid concentrations or with the positive or negative control items. In the absence of S9-mix no toxicity was observed at any of the dose levels tested. In the presence of S9-mix no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 2000 µg/mL not enough cytotoxicity was observed (31%), whereas the next higher concentration of 2262 µg/mL was too toxic for scoring (89%).

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

Without and with S9-mix : 250, 1000, 1600, 1700, 1800, 1900, 2000 and 2050¹⁾ µg/mL culture medium (3 h exposure time, 24 h fixation time).

¹⁾ The test item precipitated in the culture medium.

Table 4 (Appendix 1) shows the mitotic index of cultures treated with various 2-Benzoylbenzoic acid concentrations or with the positive or negative control items.

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

Without S9-mix : 250, 1900 and 2050 µg/mL culture medium (3 h exposure time, 24 h fixation time).

With S9-mix : 250, 1800 and 2000 µg/mL culture medium (3 h exposure time, 24 h fixation time).

In the absence and presence of S9-mix, 2-Benzoylbenzoic acid induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps wereincluded and excluded. In addition, the increases observed where above the historical controldatabase in the absence and presence of S9-mix and a significant trend (p=0.006) wasobserved in the presence of S9-mix. (Appendix 1; Table 5 - 6).

It was noted that 2-Benzoylbenzoic acid increased the number of polyploid cells and cells with endoreduplicated chromosomes in the absence of S9-mix. In the presence of S9-mix 2-Benzoylbenzoic acid 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 2-Benzoylbenzoic acid, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to 2-Benzoylbenzoic acid 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 : 125, 250, 500, 650, 800 and 1000 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 2-Benzoylbenzoic acid 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:   125, 500 and 650 µg/mL culture medium (24 h exposure time, 24 h fixation time).

125, 800 and 1000 µg/mL culture medium (48 h exposure time, 48 h fixation time).

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

2-Benzoylbenzoic acid did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Evaluation of the Results

The ability of 2-Benzoylbenzoic acid to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on the solubility of the test item in the culture medium or on toxicity, inhibition of the mitotic index of about 50% or greater.

The mitotic indices of cultures treated with various 2-Benzoylbenzoic acid 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 the test item 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.

At the 3 h exposure time in the absence and presence of S9-mix, 2-Benzoylbenzoic acid induced a statistically significant increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. In addition, the increases observed where above the 95% control limits of the distribution of the historical negative control database in the absence and presence of S9-mix and a significant trend was observed in the presence of S9-mix.

At the 24 h continuous exposure time and 48 h continuous exposure time, 2-Benzoylbenzoic acid did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

It was noted that 2-Benzoylbenzoic acid increased the number of polyploid cells and cells with endoreduplicated chromosomes at the 3 h exposure time in the absence of S9-mix. This may indicate that 2-Benzoylbenzoic acid has the potential to disturb mitotic processes and cell cycle progression. No effects of 2-Benzoylbenzoic acid on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the presence of S9-mix and in the absence of S9-mix at prolonged exposure times.

Applicant's summary and conclusion

Conclusions:

In conclusion, this test is valid and 2-Benzoylbenzoic acid is clastogenic in human lymphocytes under the experimental conditions described in this report. 2-Benzoylbenzoic acid may have the potential to disturb mitotic processes and cell cycle progression.

Executive summary:

The objective of this study was to evaluate 2-Benzoylbenzoic acid 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 2-Benzoylbenzoic acid was tested in two independent experiments.

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

The 2-Benzoylbenzoic acid was an off-white powder with a purity of 98.8%. The vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, 2-Benzoylbenzoic acid was tested up to 2050 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence of 1.8% (v/v) S9-mix. The test item precipitated in the culture medium at this dose level. 2-Benzoylbenzoic acid was tested up to 2000 µg/mL for a 3 h exposure time with a 24 h fixation time in the presence of 1.8% (v/v) S9-mix. Appropriate toxicity was reached at this dose level.

In the second cytogenetic assay, 2-Benzoylbenzoic acid was tested up to 650 µg/mL for a 24 h continuous exposure time with a 24 h fixation time. Appropriate toxicity was reached at this dose level. 2-Benzoylbenzoic acid was tested up to 1000 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. The test item precipitated in the culture medium at this dose level.

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.

At the 3 h exposure time in the absence and presence of S9-mix, 2-Benzoylbenzoic acid induced a statistically significant increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. In addition, the increases observed where above the 95% control limits of the distribution of the historical negative control database in the absence and presence of S9-mix and a significant trend was observed in the presence of S9-mix.

At the 24 h continuous exposure time and 48 h continuous exposure time, 2-Benzoylbenzoic acid did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

It was noted that 2-Benzoylbenzoic acid increased the number of polyploid cells and cells with endoreduplicated chromosomes at the 3 h exposure time in the absence of S9-mix. This may indicate that 2-Benzoylbenzoic acid has the potential to disturb mitotic processes and cell cycle progression. No effects of 2-Benzoylbenzoic acid on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the presence of S9-mix and in the absence of S9-mix at prolonged exposure times.

In conclusion, this test is valid and 2-Benzoylbenzoic acid is clastogenic in human lymphocytes under the experimental conditions described in this report. 2-Benzoylbenzoic acid may have the potential to disturb mitotic processes and cell cycle progression.