Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March 2022 to XX 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Official Journal L 142, 31/05/2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

EC Number:

219-348-1

EC Name:

Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

Cas Number:

2421-28-5

Molecular formula:

C17H6O7

IUPAC Name:

5-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyl)-1,3-dihydro-2-benzofuran-1,3-dione

Test material form:

other: solid flakes, powder

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (Hprt) enzyme locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

K1

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO K1 Chinese hamster ovary cells were purchased from American Type Culture Collection (ATCC).
- Suitability of cells: CHO cell line is selected to be used in the study as it is one of the preferred cell lines for in vitro Mammalian Cell Gene Mutation Test based on the OECD No. 476 guideline, and it was also used in the method validation study of the Test Facility.

For cell lines:
- Absence of Mycoplasma contamination: Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
- Periodically ‘cleansed’ of spontaneous mutants: Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 08 April 2018. Cells were stored as frozen stocks in a liquid nitrogen tank.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium (“culture medium”) and incubated at 37°C (± 0.5°C) in a humidified atmosphere (5± 0.3% CO2 in air).

When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.

Four types of Ham's F12 medium were prepared as follows:
F12-1: 1 % v/v Foetal bovine serum (FBS, heat inactivated); 0.01 mL/mL L-Glutamine; 0.01 mL/mL Antibiotic-Antimycotic solution*
F12-5: 5 % v/v Foetal bovine serum (FBS, heat inactivated); 0.01 mL/mL L-Glutamine; 0.01 mL/mL Antibiotic-Antimycotic solution*
F12-10: 10 % v/v Foetal bovine serum (FBS, heat inactivated); 0.01 mL/mL L-Glutamine; 0.01 mL/mL Antibiotic-Antimycotic solution*
F12-12-SEL**: 10 % v/v Foetal bovine serum (FBS, heat inactivated); 0.01 mL/mL L-Glutamine; 0.01 mL/mL Antibiotic-Antimycotic solution*

* Standard content of the antibiotic-antimycotic solution is 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B.
** Hypoxanthine-free Ham’s F-12 medium was used for preparation of the selection culture medium

Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation).
After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium and incubated for approximately 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL (if possible). Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

Treatment medium for the 24-hour treatment contained 5% (v/v) serum (F12-5).
After the 24 hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with F12-10 medium and once with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL (if possible). Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

Plating for Survival: Following adjustment of the cultures to 2x10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium. Five mL suspension (200 cells/dish) per each culture were plated into 3 parallel dishes (diameter was approx. 60 mm). The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.
Expression period: Cultures were maintained in dishes for 7 days, during which time the HPRT-mutation was expressed. During this expression period, the cultures were sub-cultured and maintained at 2x10E5 cells/dish twice (whenever possible) (on Days 1, 3, 6 and 8) to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell density was determined by haemocytometer and cells were plated for viability and for selection of the mutant phenotype.

Plating for Viability: At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL, then further diluted to 40 cells/mL using F12-10 medium. Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.

Plating for selection of the mutant phenotype (6-TG resistance): At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approx. 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 µg/mL.
6-thioguanine (6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 µg/mL) to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of the Test Facility according to Ames et al. and Maron and Ames.
Male Wistar rats (423-747 g, animals were 13-26 weeks old) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation date of the induction of liver enzymes used for preparation S9 used in this study was 23 August 2021 (Charles River Laboratories Hungary code: E13644).
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 26 August 2021.
- method of preparation of S9 mix: The S9-mix was prepared as follows:
HEPES* Concentration of the stock solution: 20 mM; Concentration in the mix: 0.2 mL/mL
KCl Concentration of the stock solution: 330 mM; Concentration in the mix: 0.1 mL/mL
MgCl2: Concentration of the stock solution: 50 mM; Concentration in the mix: 0.1 mL/mL
NADP** Concentration of the stock solution: 40 mM; Concentration in the mix: 0.1 mL/mL
D-Glucose 6 phosphate (Monosodium salt) Concentration of the stock solution: 50 mM; Concentration in the mix: 0.1 mL/mL
F12-10Concentration of the stock solution: /; Concentration in the mix: 0.1 mL/mL
S9 fraction Concentration of the stock solution: /; Concentration in the mix: 0.3 mL/mL
*HEPES = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**NADP= β-Nicotinamide-adenine dinucleotide-phosphate
Prior to addition to the culture medium the S9-mix was kept in an ice bath
- concentration or volume of S9 mix and S9 in the final culture medium: For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of the test Facility. The protein concentration of the S9 fraction used in the study was determined to be 26.1 g/L. The sterility of the preparation was confirmed.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene), that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1
5-hour treatment in the presence of S9-mix:
2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

5-hour treatment in the absence of S9-mix:
2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

Assay 2
5-hour treatment in the presence of S9-mix:
2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

24-hour treatment in the absence of S9-mix:
2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (distilled water)

- Justification for choice of solvent/vehicle:
A trial formulation was performed at the Test Facility using distilled water. Homogeneous pale-yellow suspension was observed at 200 mg/mL concentration. Therefore, distilled water was used as vehicle (solvent) for the test item. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.

- Justification for percentage of solvent in the final culture medium: not applicable (Distilled water was used as the vehicle).

Controls

Untreated negative controls:

yes

Remarks:

to demonstrate that the selected vehicle (solvent) had no mutagenic effects.

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

yes

Positive controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO) was used as vehicle of the positive control materials.

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
Duplicate cultures were used for each treatment. Plating for Survival and Plating for Viability was done in triplicate. Plating for selection of mutant phenotype was done in 5 parallels per sample.
- Number of independent experiments: The study included one preliminary experiment for dose range finding and two main tests.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): At least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No preincubation period
- Exposure duration/duration of treatment:
Assay 1 (with and without metabolic activation) and Assay 2 (with metabolic activation): 5 h treatment
Assay 2 (without metabolic activation): 24 h treatment
- Harvest time after the end of treatment (sampling/recovery times):
5 h treatment: 19 h incubation after treatment
24 h treatment: 0 h incubation after treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
Cultures were maintained in dishes for 7 days, during which time the HPRT-mutation was expressed.
- Selection time (if incubation with a selective agent): 7 days
At the end of the expression period (Day 8), 6-thioguanine (abbreviation: 6-TG) was added to the dishes to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.
- Fixation time (start of exposure up to fixation or harvest of cells):
Survival: 6 days (5 h treatment, 19 h incubation, 5 days growth or 24 h treatment , 5 days growth)
Viability: 13 days (5 h treatment, 19 h incubation, 7 days expression, 5 days viability or 24 h treatment, 7 days expression, 5 days viability)
Mutant selection:15 days (5 h treatment, 19 h incubation, 7 days expression, 7 days mutant selection or 24 h treatment, 7 days expression, 7 days mutant selection)
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 6-thioguanine (final volume: 10 mL, final 6-TG concentration: 10 µg/mL). Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
*Plating for Viability: At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL, then further diluted to 40 cells/mL using F12-10 medium.
Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.
*Plating for selection of the mutant phenotype (6-TG resistance): At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approx. 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 µg/mL
6-thioguanine (abbreviation: 6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 µg/mL) to determine mutation frequency. Dishes were incubated at 37°C ±0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.
After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival (RS): Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells.
The mutation frequencies were statistically analysed.

Evaluation criteria:

The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the general historical control data.
2. The positive control chemicals induced a statistically significant increase in mutant frequency and should be within the historical data for positive controls.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8; under these conditions an adequate number of cells are analysable.
4. At least four test item concentrations in duplicate cultures were presented.

The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The general historical control range is considered when deciding if the result is positive.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary).
According to the relevant OECD 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

Statistics:

The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Assay 1: 5 h treatment; An evaluation was made using data of five of the lower concentrations based on the acceptable cytotoxicity data.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Assay 1: 5 h treatment; An evaluation was made using data of six of the lower concentrations based on the acceptable cytotoxicity data.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Assay 2: 5 h treatment; An evaluation was made using data of five of the lower concentrations based on the acceptable cytotoxicity data.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Assay2: 24 h treatment; An evaluation was made using data of five of the lower concentrations based on the acceptable cytotoxicity data.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: In Assay 1 and Assay 2, there were no large changes in pH after treatment in any cases.
- Data on osmolality: : In Assay 1 and Assay 2, there were no large changes in osmolality after treatment in any cases.
- Precipitation and time of the determination: In Assay 1, insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation at the concentration range of 2000-500 µg/mL.
In Assay 2, insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation at the concentration range of 2000 - 500 and/or 250 µg/mL.

RANGE-FINDING/SCREENING STUDIES (if applicable): Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix were performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 µg/mL (up to the maximum recommended concentration).
Insolubility was detected in the preliminary experiment in the final treatment medium at the end of the treatment with and without metabolic activation. The concentrations selected for the main experiments were based on results of the performed Preliminary Toxicity Tests according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit and the recommended maximum concentration). At least eight test item concentrations were selected for the main experiments.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays and the observed values were in the expected range (5-20 x 10E-6) as shown in the OECD No. 476 guideline.
The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays.
The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
In Assay 1 in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 10%, 22% and 58%, respectively).
In Assay 1 in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 21%, 24% and 55%, respectively).
In Assay 2 in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 9%, 32% and 49%, respectively).
In Assay 1 in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 28%, 29% and 41%, respectively).
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: See under “Any other information on results incl. tables”

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
For the 5-hour treatments, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium and incubated for approximately 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).

After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL (if possible). Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

For the 24-hour treatment, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for approximately 24 hours before treatment at 37°C ± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5% (v/v) serum (F12-5). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 24 hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with F12-10 medium and once with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL (if possible). Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

Duplicate cultures were used for each treatment. Solubility of the test item in the cultures was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment.
o Number of cells plated in selective and non-selective medium
*non-selective medium:
Following adjustment of the cultures to 2x10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium. Five mL suspension (200 cells/dish) per each culture were plated into 3 parallel dishes (diameter was approx. 60 mm). The dishes were incubated at 37°C ±0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing (Plating for Survival).
Cultures were maintained in dishes for 7 days, during which time the HPRT-mutation was expressed. During this expression period, the cultures were sub-cultured and maintained at 2x10E5 cells/dish twice (whenever possible) (on Days 1, 3, 6 and 8) to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell density was determined by haemocytometer and cells were plated for viability and for selection of the mutant phenotype.
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL, then further diluted to 40 cells/mL using F12-10 medium. Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing (Plating for Viability).
*selective medium:
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approx. 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 µg/mL 6-thioguanine (6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 µg/mL) to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing (Plating for selection of the mutant phenotype (6-TG resistance).
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See under “Any other information on results incl. tables”

HISTORICAL CONTROL DATA See under “Any other information on results incl. tables”

Any other information on results incl. tables

Table 1: Survival Results of Assay 1

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)	Relative Survival (%) on plates
+	5	A1	2000 µg/mL	-	-	-
			1500 µg/mL	-	-	-
			1250 µg/mL	-	-	-
			1000 µg/mL	109	0.091	10
			750 µg/mL	234	0.195	22
			500 µg/mL	613	0.511	58
			250 µg/mL	777	0.648	73
			125 µg/mL	909	0.758	86
			6.25 µg/mL	964	0.803	91
			Negative control	1061	0.884	100
			Negative control for DMBA (DMSO)	924	0.770	87
			Untreated control	1136	0.947	107
			Positive control (DMBA)	3	0.003	0
-	5	A1	2000 µg/mL	-	-	-
			1500 µg/mL	-	-	-
			1250 µg/mL	-	-	-
			1000 µg/mL	268	0.223	21
			750 µg/mL	307	0.256	24
			500 µg/mL	702	0.585	55
			250 µg/mL	1333	1.111	103
			125 µg/mL	1263	1.053	98
			6.25 µg/mL	1229	1.024	95
			Negative control	1288	1.073	100
			Negative control for EMS (DMSO)	1272	1.060	99
			Untreated control	1252	1.043	97
			Positive control (EMS)	1012	0.843	79

A1 = Assay 1

+ = in the presence of S9-mix                                DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                   EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control = 1% (v/v) distilled water

DMSO = Dimethyl sulfoxide

The first three concentrations could not be plated for survival testing due to excessive cytotoxicity.

 

 

Table 2: Survival Results of Assay 2

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)	Relative Survival (%) on plates
+	5	A2	2000 µg/mL	-	-	-
			1500 µg/mL	-	-	-
			1250 µg/mL	-	-	-
			1000 µg/mL	135	0.113	9
			750 µg/mL	473	0.394	32
			500 µg/mL	730	0.608	49
			250 µg/mL	964	0.803	65
			125 µg/mL	1322	1.102	89
			6.25 µg/mL	1368	1.140	92
			Negative control	1479	1.233	100
			Negative control for DMBA (DMSO)	1357	1.131	92
			Untreated control	1473	1.228	100
			Positive control (DMBA)	67	0.056	5
-	24	A2	2000 µg/mL	-	-	-
			1500 µg/mL	-	-	-
			1250 µg/mL	-	-	-
			1000 µg/mL	178	0.297	28
			750 µg/mL	369	0.308	29
			500 µg/mL	527	0.439	41
			250 µg/mL	1201	1.001	93
			125 µg/mL	1352	1.127	105
			6.25 µg/mL	1286	1.072	100
			Negative control	1289	1.074	100
			Negative control for EMS (DMSO)	1256	1.047	97
			Untreated control	1248	1.040	97
			Positive control (EMS)	430	0.358	33

A2 = Assay 2

+ = in the presence of S9-mix                             DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                               EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control = 1% (v/v) distilled water

DMSO = Dimethyl sulfoxide

The first three concentrations and the culture B of the 1000 µg/mL concentration could not be plated for survival testing due to excessive cytotoxicity.

 

 

Table 3: Viability Results of Assay 1

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)
+	5	A1	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	-	-
			750 µg/mL	1251	1.043
			500 µg/mL	1087	0.906
			250 µg/mL	1191	0.993
			125 µg/mL	1221	1.018
			6.25 µg/mL	1268	1.057
			Negative control	1126	0.938
			Negative control for DMBA (DMSO)	1049	0.874
			Untreated control	1080	0.900
			Positive control (DMBA)	708	1.180
-	5	A1	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	1242	1.127
			750 µg/mL	1353	1.128
			500 µg/mL	1089	0.908
			250 µg/mL	896	0.747
			125 µg/mL	1029	0.858
			6.25 µg/mL	1211	1.009
			Negative control	1164	0.970
			Negative control for EMS (DMSO)	1243	1.036
			Untreated control	1246	1.038
			Positive control (EMS)	826	0.688

A1 = Assay 1

+ = in the presence of S9-mix                             DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control = 1% (v/v) distilled water

DMSO = Dimethyl sulfoxide

The first three or four concentrations could not be plated for viability testing due to excessive cytotoxicity.

 

 

Table 4: Viability Results of Assay 2

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)
+	5	A2	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	-	-
			750 µg/mL	1231	1.026
			500 µg/mL	1288	1.073
			250 µg/mL	1129	0.941
			125 µg/mL	1128	0.940
			6.25 µg/mL	1148	0.957
			Negative control	1186	0.988
			Negative control for DMBA (DMSO)	1077	0.898
			Untreated control	1242	1.035
			Positive control (DMBA)	1023	0.853
-	24	A2	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	-	-
			750 µg/mL	1195	0.996
			500 µg/mL	1083	0.903
			250 µg/mL	1111	0.926
			125 µg/mL	1136	0.947
			6.25 µg/mL	1127	0.939
			Negative control	1135	0.946
			Negative control for EMS (DMSO)	1061	0.884
			Untreated control	1253	1.044
			Positive control (EMS)	312	0.260

A2 = Assay 2

+ = in the presence of S9-mix                             DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                               EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control = 1% (v/v) distilled water

DMSO = Dimethyl sulfoxide

The first four concentrations could not be plated for viability testing due to excessive cytotoxicity.

 

 

Table 5: Mutagenicity Results of Assay 1

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Mutant frequency
+	5	A1	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	-	-
			750 µg/mL	27	6.5
			500 µg/mL	29	8.0
			250 µg/mL	33	7.5
			125 µg/mL	33	8.1
			6.25 µg/mL	32	6.0
			Negative control	40	10.6
			Negative control for DMBA (DMSO)	40	11.7
			Untreated control	39	10.9
			Positive control (DMBA)	322	136.4**
-	5	A1	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	11	2.4
			750 µg/mL	15	3.3
			500 µg/mL	29	8.0
			250 µg/mL	32	13.3
			125 µg/mL	35	10.2
			6.25 µg/mL	42	10.2
			Negative control	28	7.2
			Negative control for EMS (DMSO)	34	8.1
			Untreated control	44	10.5
			Positive control (EMS)	1151	418.1**

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control

 

 

A1 = Assay 1

+ = in the presence of S9-mix                             DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control = 1% (v/v) distilled water

DMSO = Dimethyl sulfoxide

 

Mutant frequencies refer to 10⁶ clonable cells.

At least five concentrations were evaluated at mutagenicity testing in both cases based on the cytotoxicity data (in bold).

The first three or four concentrations and culture A of the positive control in the presence of S9-mix could not be plated for mutagenicity testing due to excessive cytotoxicity.

 

 

Table 6: Mutagenicity Results of Assay 2

S9 mix	Treatment period (hours)	Study phase	Test item or control concentration	Total number of colonies	Mutant frequency
+	5	A2	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	-	-
			750 µg/mL	15	3.6
			500 µg/mL	16	3.8
			250 µg/mL	13	3.2
			125 µg/mL	18	4.8
			6.25 µg/mL	26	8.4
			Negative control	27	7.1
			Negative control for DMBA (DMSO)	25	6.8
			Untreated control	21	5.1
			Positive control (DMBA)	1357	400.2**
-	24	A2	2000 µg/mL	-	-
			1500 µg/mL	-	-
			1250 µg/mL	-	-
			1000 µg/mL	-	-
			750 µg/mL	11	2.7
			500 µg/mL	12	3.3
			250 µg/mL	25	6.1
			125 µg/mL	10	2.7
			6.25 µg/mL	11	3.7
			Negative control	17	4.5
			Negative control for EMS (DMSO)	25	7.1
			Untreated control	21	5.1
			Positive control (EMS)	601	613.4**

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control

 

A2 = Assay 2

+ = in the presence of S9-mix                             DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL

- = in the absence of S9-mix                                EMS = Ethyl methanesulfonate, 0.4 µL/mL

Negative (vehicle) control = 1% (v/v) distilled water

DMSO = Dimethyl sulfoxide

 

Mutant frequencies refer to 10⁶ clonable cells.

Five concentrations were evaluated at mutagenicity testing in both cases based on the cytotoxicity data (in bold).

The first four concentrations could not be plated for mutagenicity testing due to excessive cytotoxicity.

 

 

Table 7: Historical Control Data

(Updated on 04 April 2022 using data of GLP studies Period of July 2010 - January 2022)

 

	Mutation frequency (Number of 6-TG resistant mutants per 10^6 clonable cells)
	Untreated control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	10.2	10.5	10.4
standard deviation	10.2	11.0	11.1
minimum	3.5	4.0	3.3
maximum	64.1	55.5	58.0
n	85	44	45
	DMSO control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	11.5	10.1	10.3
standard deviation	11.7	8.5	9.7
minimum	3.8	4.6	4.1
maximum	57.3	47.4	48.5
n	87	44	45
	Distilled water / Water based vehicle control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	7.4	7.5	7.7
standard deviation	2.9	2.6	4.5
minimum	3.3	5.0	4.0
maximum	15.8	13.7	20.1
n	30	17	16
	Positive controls
	DMBA	EMS	EMS
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	621.2	427.9	861.0
standard deviation	397.0	125.2	437.9
minimum	132.9	174.3	190.8
maximum	2119.4	922.2	2449.8
n	85	45	44

 

DMSO = Dimethyl sulfoxide

DMBA = 7,12-Dimethylbenz[a]anthracene

EMS = Ethyl methanesulfonate

S9+ = in the presence of S9-mix

S9- = in the absence of S9-mix

Applicant's summary and conclusion

Conclusions:

In conclusion, no mutagenic effect of Benzophenone-3,3':4,4'-tetracarboxylic dianhydride (BTDA) was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of Benzophenone-3,3':4,4'-tetracarboxylic dianhydride (BTDA) to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice.

 

Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

 

Assay 1

5-hour treatment in the presence of S9-mix:

2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

 

5-hour treatment in the absence of S9-mix:

2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

 

Assay 2

5-hour treatment in the presence of S9-mix:

2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

 

24-hour treatment in the absence of S9-mix:

2000, 1500, 1250, 1000, 750, 500, 250, 125 and 62.5 µg/mL.

 

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony-forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

 

In Assays 1 and 2, insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation at the concentration range of 2000 - 500 and/or 250 µg/mL. There were no large changes in pH and osmolality after treatment in any cases.

 

In the Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 10%, 22% and 58%, respectively*). An evaluation was made using data of five of the lower concentrations based on the acceptable cytotoxicity data. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R²= 0.049)). This experiment is considered to be negative.

 

*Note: The first three concentrations could not be plated for survival testing due to excessive cytotoxicity.

 

In the Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 21%, 24% and 55%, respectively*). An evaluation was made using data of six of the lower concentrations based on the acceptable cytotoxicity data. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R²= 0.574)). This experiment is considered to be negative.

 

*Note: The first three concentrations could not be plated for survival testing due to excessive cytotoxicity.

 

In the Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 9%, 32% and 49%, respectively*). An evaluation was made using data of five of the lower concentrations based on the acceptable cytotoxicity data. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R²= 0.670)). This experiment is considered to be negative. Moreover, it confirmed the result of the Assay 1.

 

*Note: The first three concentrations could not be plated for survival testing due to excessive cytotoxicity.

 

In the Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (1000, 750 and 500 µg/mL concentrations showed, with a relative survival of 28%, 29% and 41%, respectively*). An evaluation was made using data of five of the lower concentrations based on the acceptable cytotoxicity data. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R²= 0.013)). This experiment is considered to be negative.

 

*Note: The first three concentrations and the culture B of the 1000 µg/mL concentration could not be plated for survival testing due to excessive cytotoxicity.

 

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least five evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

 

In conclusion, no mutagenic effect of Benzophenone-3,3':4,4'-tetracarboxylic dianhydride (BTDA) was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.