Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the in vitro Ames, micronucleus and HPRT assays, the test substance is not considered to be genotoxic.

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:

From November 05, 2012 to November 29, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 5307; Purity: >99.9%

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2µg/plate for WP2uvrA, 3µg/plate for TA100, 5µg/plate for TA 1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

80µg/plate for TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2µg/plate for TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-amino anthracene

Remarks:

10µg/plate for WP2uvrA, 1µg/plate for TA100, 2µg/plate for TA1535 and TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

5µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 48 h at 37°C
- Exposure duration: 20 min with shaking

NUMBER OF REPLICATS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: microscopic evaluation of thinning

EXPERIMENT I - Plate incomporation method

Eight concentrations of the test subsance 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate were assayed in tripliate against each tester strain, using the direct plate incomporation method.

Without metabolic activation:
0.1 mL of appropriate concentration of the test substance, vehicle or appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45°C) containing 1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were than mixed and overlayed onto a Vogel-Boner agar plate. Negative (untreated) controles were also performed on the same day as the mutation test. Each concentration of the test substance,m appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With metabolic activation:
Same procedure was applied as for study without metabolic activation except that following the addition of the test substance formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media intead of phosphate buffer.

Incubation and scoring:
All of the plates were incubated at 37 °C ± 3°C for approximately 48 h and scored for the presence revertant colonies using an automated colony counting system. The plates were viewd microscopically for evidence of thinning (toxicology).

EXPERIMENT II - test for mutagenicity - Pre-Incubation Method

As experiment I was deemed negative. Experiment 2 was performed using the pre-incubation method in the presence and absece of metabolic ativation.

Without metabolic activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test substance formulation, vehicle or appropriate positive control were incubated at 37 ± 3°C for 20 min (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent planting onto a Vogel-Boner plates. Negative (untreated) controles were also performed on the same day as the mutation test. All testing for this experiment was performed in triplicates.

With metabolic activation:
Same procedure was applied as for study without metabolic activation except that following the addition of the test substance formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media intead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 min (with shaking) and addition of amino-acid supllemented media. All testing for this experiment was performed in triplicate.

Incubation and scoring:
All of the plates were incubated at 37 ± 3°C for approximately 48 h and scored for the presence revertaant colonies using an automated colony counting system. The plates were viewd microscopically for evidence of thinning (toxicology).

Evaluation criteria:

There are several criteria for determining positive result. Any one or all of the following can be used to determine overall result of the study:
1. Dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statystical analysis of data as determined by UKEMS (Mahon et al. 1989).
5. Fold increase greater than two times the concurent solvent control for any tester strain (especially if acompanied by out-of-historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.Although most experiments will give clear positive or negative results, is some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

INFORMATION ON CYTOTOXICITY: cytotoxicity was observed at 5000 µg/plate with and without metabolic activation for all strains.

In Experiment I (plate incorporation method) the test substance induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the tester strais from 1500 µg/plate (Sallmonella strains) and at 5000 µg/plate (Escherichia Coli strain WP2uvrA) both in the presence and absence of metabolic activation. Consequently the same maximum dose level was used in the second mutation test of Escherichia Coli and the toxic limit of the test substance was selected for the Salmonella typhimurium strains. The toxic response of the test substance in Experiment II (pre-incubation method) closely followed the results of the first experiment with weakened lawns noted to the Sallmonella strains from 1500 µg/plate and to Escherichia Coli strain WP2uvrA 5000 µg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either presence or absence of the S9-mix.

There were no significant increases in the frequency of relevant colonies recorded for any of the bacterial strain, with any dose of the test substance, either with or withourt metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency revertant colonies thus confirming the activity of the S9-mix and sensitivity of the bacterial strains. Results for the negative controls are also considered to be acceptable.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

Remarks on result:

other: not mutagenic

Conclusions:

Under the study conditions, the test substance was not found to be mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance PBBA, according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA in the Ames plate incorporation and pre-incubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment I was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels and an expanded dose range were selected in Experiment 2 in order to achieve both non-toxic dose levels and the toxic limit of the test substance. Results for the positive and negative controls were in line with the historical laboratory data. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation in both the experiment. Cytotoxicity was observed at 5000 µg/plate with and without metabolic activation for all strain. Under the study conditions of the in vitro Ames test, the test substance was considered to be non-mutagenic to S typhimurium strains, both with and without metabolic activation (Thompson, 2013).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 16, 2013 to February 10, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch No.: 5307; Purity: 99.9%

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The maximum dose level of the test substance was 3020 µg/mL, which was calculated to be equivalent to 10 mM, the maximum recommended dose level.

Vehicle / solvent:

The test substance was insoluble in culture media at 30.20 mg/mL but was soluble in DMSO at 302 mg/mL in solubility checks performed in-house. There was no significant change in pH when the test substance was doesed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

MICROSOMAL ENZYME FRACTION

The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally for 3 d prior to preparation on Day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15 M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant were frozen and stored approximately -196°C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

The S9-mix was prepared prior to the dosing of the test cultures and contained S9 fraction (20% (v/v)). MgCl2, (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), and NADP (5 mM). The final concentration of S9, when dosed at a 10 % volume of S9-mixinto culture media, was 2%.

TEST PROCEDURE

Culture Conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
- 9.05 mL MEM, 10% FBS
- 0.1 mL Li-heparin
- 0.1 mL phytohaemagglutinin
- 0.75 mL heparinized whole blood

MAIN TESTS

With metabolic activation (S9):

After 48 h incubation at 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved and replaced with the required volume of MEM (including serum) and dosed with 0.1 mL of the appropriate solution of vehicle control or test substance was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0% of 20% S9-mix (i.e. 2% final concentration of S9 in final co-factors) was added to the cultures of the preliminary toxicity test and of Experiment I. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 h at approximately 37°C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 28 h.

Without metabolic activation (S9):

After 48 h incubation at 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved. The cells were then suspended in required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate solution of vehicle control, test substance or 0.1 mL of positive control. The nominal total volume for each culture was a 10 mL.
After 4 hours at approximately 37°C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 28 h.
In the Experiment II, in the absence of metabolic activation, the exposure was continuous for 24 h. Therefore, when the cultures were established the culture volume was nominal 9.9 mL. After approximately 48 h incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test substance dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 h, the tues and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then incubated for a further 28 h.
The preliminary toxicity test was performed using the exposure conditions as described for Experiment I and for Experiment II.

Experiment I:
- 4-h exposure to the test substance without S9-mix followed by 28 h incubation periodin treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
- 4-h exposure to the test substance withmS9-mix (2%), followed by 28 h incubation periodin treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test substance used was: 45, 90, 180, 240, 300, 360 µg/mL.
Experiment II:
- 24-h continuous exposure to the test substance without S9-mix followed by 28 h incubation periodin treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test substance was 22.5, 45, 90, 120, 180, 240, 300 µg/mL.

PRELIMINARY TEST

Preliminary Toxicity test: three exposure groups were used same as above-mentioned for the main tests but with the test substance concentration range within: 11.80 to 3020 µg/mL.
Pararell flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test substance precipitate observations could be made. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, approperiate dose levels were selected for the evaluation of the frequnecy of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI . The CBPI data were used to estimate test substance toxicity and for selection of the dose levels for the experiments of the main test.

Preparation of Microscope slides:
The lymphocytes were re-suspended in several mL of fresh flexative before centrafugation and re-suspension in small amount of flexative. Several drops of this suspension ere droped onto celan, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

CELL HARVEST

At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4°C prior to slide making.

Evaluation criteria:

Cytokinesis Block Proliferation Index:

Cytotoxicity = 100{(CBPIT - 1)/(CBPI C - 1)}

Where:
CBPI = No. (mononucleate cells + 2 x No. binucleate cells) + (3 x No. multinucleate cells) / Total number of cells

And:
T = test chemical treatment culture
C = vehicle control culture

Positive control: All the positive control chemicals must induce positive responses (p<0.01). Acceptable positive responses demonstrate validity of the experiment and the integrity of S9-mix.
Negative control: The frequency of binucleate cells with micronuclei in the vehicle control cultures will ormally be within laboratory historical control data range.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in frequency of cells with aberrations which was reproducible.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TEST

Aggregated precipitate of the test substance was observed in the parallel blood-free cultures at the end of exposure, at above 1510 µg/mL, in the 4-hour exposure group in the absence of S9 and at 3020 µg/mL in the 4-h exposure group in the presence of S9 and in the 24-h continuous exposure group.

Haemolysis was observed following exposure to the test substance at and above 755 µg/mL in both 4-h exposure groups in the presence and absence of S9, and at and above 377.5 µg/mL in the 24-h continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultured showed that binucleate cells were present at up to 377.5 µg/mL in the 4-h exposure groups, both in the presence and absence of metabolic activation (S9). The maximum dose with binucleate cells present in the 24-h continuous exposure was 188.75 µg/mL.

The selection of maximum dose level was based on toxicity and was 360 µg/mL for the 4-h exposure groups in the presence and absence of S9, and was 300 µg/mL for the 24-h exposure group used in Experiment 2.

EXPERIMENT I – micronucleus test

The qualitative assessment of the slides indicated that the toxicity was marginally greater than that observed in the preliminary test ad that there were binucleate cells suitable for scoring up to 300 µg/mL in both exposure groups.

The CBPI data confirm the qualitative observations in that dose-related inhibition of CBPI was observed

No precipitate was observed at the end of the exposure period in either exposure group. The dose level of 360 µg/mL in both exposure groups was toxic with no binucleate cells suitable for scoring.

There were statistically significant increases in the numbers of micronuclei at 300 µg/mL in both exposure groups. The observed increases were not part of a true dose-related response, only occurring at the upper dose level where marked toxicity was observed.

EXPERIMENT II

A cloudy precipitate was observed at the end of exposure at 300 µg/mL and haemolysis was also seen at this dose level.

The CBPI data confirm the qualitative observations in that dose-related inhibition of CBPI was observed, and that 62% and 44% inhibition of cell proliferation was achievedat 180 µg/mL and 120 µg/m respectively. The dose levels with 240 µg/mL with 77 % inhibition of cell proliferation and 300 µg/mL with no binucleate cells were both too toxic for scoring.

The test substance did not induce any statistically significant increases in the frequency of cells with micronuclei. The maximum dose 300 µg/mL achieved greater than optimum toxicity with 62% inhibition of cell proliferation and demonstrated no increases in the frequency of cells with micronuclei, confirming that the small increases seen in 4-h exposures of experiment I were of no toxicological significance.

Remarks on result:

other: not clastogenic

Conclusions:

Under the study conditions, the test substance was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

A study was conducted to determine the clastogenic and aneugenic potential of the test substance PBBA, according to OECD Guideline 487, in compliance with GLP. The dose levels used in the main experiments were selected using data from the preliminary toxicity test. Three independent exposures were performed. Experiment I used a 4 h exposures in the presence and absence of a standard metabolizing system (2% S9 mix). The following concentrations were evaluated: 45, 90, 180, 240, 300, 360 µg/mL. Experiment II, used a 24 h exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of experiment I. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B. Examined concentrations in this experiment were as follows: 22.5, 45, 90, 120, 180, 240, 300 µg/mL. The metabolic activation system has shown to be functional as results for the positive and negative control were as expected. No biologically relevant increase in the number of binucleated cells containing micronuclei was observed in both experiments (with and without metabolic activation), using a dose range that included approximately 50% reduction in CBPI or greater. Under the study conditions of the in vitro micronucleus assay, the test substance was considered to be non-clastogenic and non-aneugenic to human lymphocytes, both with and without metabolic activation (Morris, 2014).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 February, 2019 to 07 May, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

See discussion under 'any other information on materials and methods incl. tables'

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

See discussion under 'any other information on materials and methods incl. tables'

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-(4-Phenylbenzoyl)benzoic acid
- Physical state: White powder
- Analytical purity: 99.6%
- Lot/batch No.: Sarex #7650
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%), Protected from light and humidity

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium with 1-10% foetal bovine serum (FBS, heat inactivated)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: No

Additional strain / cell type characteristics:

other:

Remarks:

Sub-line (K1) of Chinese hamster ovary cell line CHO

Metabolic activation:

with and without

Metabolic activation system:

The post-mitochondrial fraction (S9) [7, 8] was prepared from rat liver by the Microbiological Laboratory of Citoxlab Hungary Ltd. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived annually.

Induction of Liver Enzymes:
Male Wistar rats (292-387 g, animals were 8 weeks old at initiation of E12790) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was performed by ascending concentration of CO2 and death was confirmed by cutting through major thoracic blood vessels. Initiation dates of the induction of liver enzymes used for preparation S9 used in this study were 05 January 2018.

Preparation of Rat Liver Homogenate S9 Fraction:
On Day 4, the rats were euthanized 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 then 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 used in this study was 08 January 2018 (Citoxlab code: E12790, Expiry date: 08 January 2020).

The sterility of the preparation was confirmed. The protein concentration was determined by colorimetric test by chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 30.45 g/L.

The biological activity in the Salmonella assay of each batch of S9 was characterized beside the 2-Aminoanthracene with another mutagen, Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately during the activity checking.

PREPARATION OF THE S9-MIX
Treatments were carried out both in the absence and presence of S9 mix. The S9-mix was prepared as follows:
HEPES*: 20 mM (Concentration of the stock solution), 0.2 mL/mL (Concentration in the mix)
KCl: 330 mM, 0.1 mL/mL
MgCl2: 50 mM, 0.1 mL/mL
NADP**: 40 mM, 0.1 mL/mL
D-Glucose-6-phosphate (Monosodium salt): 50 mM, 0.1 mL/mL
F12-10:-, 0.1 mL/mL,
S9 fraction: -, 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.
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%.

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 µg/mL (5-h treatment)
with S9 mix: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 µg/mL (5-h treatment)

Experiment III:
without S9 mix: 175, 160, 150, 140, 125, 62.5, 31.25 and 15.625 µg/mL (24-h treatment)
with S9 mix: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 µg/mL (5-h treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (1% (v/v))

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethyl methanesulfonate: 0.4 µL/mL (final concentration)

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

7,12-Dimethylbenz[a]anthracene: 15 µg/mL (final concentration)

Details on test system and experimental conditions:

FORMULATION:
Based on the results of a short solubility test, distilled water was not a suitable solvent for the test, however the test substance was soluble at 200 mg/mL concentration in Dimethyl sulfoxide (DMSO) which is an accepted solvent for this test. Therefore, it was selected for vehicle of the study. This vehicle (solvent) was compatible with the survival of the mammalian cells and the metabolic activation system. The highest test concentration in the preliminary test was 2000 μg/mL.
The test substance was formulated in the selected vehicle (solvent) to provide a suitably concentrated stock solution as follows: the necessary amount of test item was weighed into a calibrated volumetric flask. Approximately 80% of the required volume of vehicle (solvent) was added and the formulation was stirred until homogeneity was reached, then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared using the selected vehicle (solvent) to prepare dosing solutions for lower doses. The vehicle (solvent) were filtered sterile using a 0.22 μm syringe filter (Supplier: Millipore, Lot No.: R7KA43538, Expiry date: August 2020 was used in the preliminary experiment and Lot No.: R7KA37756, Expiry date: August 2020 was used in the Assay 1 and R8DA52451, Expiry date: April 2021) before the preparation of the dosing formulations in each case. The stock solutions as well as all dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.
Analytical determination of the test substance concentration, stability and homogeneity was not performed because of the character and short period of the study.

NEGATIVE AND POSITIVE CONTROLS
Negative (vehicle) and positive controls were included in the experiments. In addition, untreated control sample was also used to demonstrate that the selected vehicle (solvent) had no mutagenic effects. Routine safety precautions for controls (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.
- Negative (Vehicle) Control: Based on the results of a short solubility test, DMSO as vehicle was suitable for the test. The negative control cultures were treated with the vehicle (solvent) only in the same way as the test item treated cultures.
- Positive Controls: Ethyl methanesulfonate, a widely used positive control in the absence of metabolic activation, was dissolved in DMSO and used at a final concentration of 0.4 μL/mL. 7,12-Dimethylbenz[a]anthracene, a mutagen that requires metabolic transformation by microsomal enzymes, was dissolved in DMSO and used as a positive control substance for the experiments with metabolic activation at a final concentration of 15 μg/mL. Positive control solutions were freshly prepared at the beginning of the experiments in the testing laboratory in a sterile hood and were filtered sterile using a 0.22 μm syringe filter before use (Supplier: Millipore, Lot No.: R8DA52451, Expiry date: April 2021).

INDICATOR CELLS:
The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased from American Type Culture Collection (ATCC). 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. Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium (“culture medium”, the content of the medium is listed in Section 5.4.) and incubated at 37oC (± 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.

TEST PROCEDURE:
Principles of dose selection (Preliminary Toxicity Test)
- Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. For the treatments in the preliminary toxicity test, a formulation of 200 mg/mL was prepared using the selected vehicle (solvent) and lower test concentrations were prepared by serial dilutions.
- The highest test concentration in the preliminary test was 2000 μg/mL (the maximal recommended concentration*).
- In the preliminary experiment, a 5-h treatment in the presence and absence of S9-mix and a 24-h treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments.
- Treatment of cell cultures was performed as described below for the main mutation assays. However, single cultures were tested and positive controls were not included. Following treatments, cell number in the samples was adjusted to 2x105 cells/mL (if possible) after counting and cells (10 mL cell suspension) were transferred to dishes for growth some additional days. After the incubation period, cell concentrations were determined using a haemocytometer on Day 4, 6 and 8 in the preliminary experiment.
- Precipitation of the test item in the final culture medium was visually examined at the beginning and end of the treatments. The pH and osmolality of the treatment medium at the end of the treatment was also determined.

Mutation Assays:
In experiment 1, 5-h treatment was performed with and without metabolic activation (in the presence and absence of S9-mix). In experiment 3, 3, 5-h treatment was performed with metabolic activation (in the presence of S9-mix) and 24-h treatment without metabolic activation (in the absence of S9-mix).
Note: Based on the observed results of the mutagenicity plates in experiment 2 with and without metabolic activation, the positive control groups did not fulfil the acceptance criteria, therefore the experiment is considered to be invalid. Furthermore, in some cases unexpected higher colony counts were observed in the test item treated groups.
An additional experiment (experiment 3) was performed to provide additional data for a fully valid study.

Treatment of the cells
- For the 5-h treatments, at least 2x106 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and incubated for about approximately 24 h 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-h 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) and test substance solution or positive control solution and their vehicles 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.
- After the 5-h 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 (10-60 mL) and incubated for 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
- After the 19-h 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 2x105 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-h treatment, at least 2x106 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and incubated for approximately 24 h 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-h treatment contained 5% serum (F12-5). A suitable volume (100 μL) of vehicle (solvent) and test substance solution or positive control solution and their vehicles 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-h incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), 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 2x105 cells/mL. 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.

Plating for survival
Following adjustment of the cultures to 2x105 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium as follows (typical ratio is shown, other dilutions using the same ratio were also acceptable):
- Survival: Initial cell conc.: 2 x 105 cells/mL (dilution: A: 0.1 mL, F12-10: 9.9 mL); Intermediate cell conc.: 2 x 103 cells/mL (dilution: B: 0.4 mL, F12-10: 19.6 mL); Final cell conc.: 40 cells/mL
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 2x105 cells/dish (if possible) twice (on Days 3 or 4, 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 4x105 cells/mL, then further diluted to 40 cells/mL using F12-10 medium.
- Viability: Initial cell conc.: 4 x 105 cells/mL (dilution: A: 0.1 mL, F12-10: 9.9 mL); Intermediate cell conc.: 4 x 103 cells/mL (dilution: B: 0.2 mL, F12-10: 19.86 mL); Final cell conc.: 40 cells/mL
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 4x105 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.

Fixation and staining of colonies
- 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.

ANALYSIS OF THE RESULTS
- Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
- The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x106 cells: 5 plates at 4x105 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 106 clonable cells.
- 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 homogeneity 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 abnormal 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).
- In the statistical analysis, negative trends were not considered significant.

Acceptance criteria
The assay was considered valid if all the following criteria are met:
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 clear increase in mutant frequency
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test item concentrations in duplicate cultures were presented.
Note: No criteria about historical data for positive controls was included in the Study Plan, but positive control values were compatible with those generated in the historical positive control data base.

Rationale for test conditions:

See above section

Evaluation criteria:

The test substance was considered to be mutagenic in this assay if the following criteria are 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.

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 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 homogeneity 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 abnormal 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). In the statistical analysis, negative trends were not considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: No insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation.

PRELIMINARY EXPERIMENT:
- Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. In the preliminary experiment, a 5-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 µg/mL.
- Cytotoxicity was detected in the preliminary experiment. The concentrations selected for the main experiments were based on the cytotoxicity data to cover the range from cytotoxicity to no or little cytotoxicity according to the relevant OECD guideline.

MUTATION ASSAYS
- In the mutation assays, cells were exposed to the test item for 5 h with and without metabolic activation system (±S9-mix) or for 24 h without metabolic activation system (-S9-mix) then the cells were plated for determination of survival and in parallel sub-cultured without test item for 7 d to allow the expression of the genetic changes (if any occurred). At the end of the expression period, cells were allowed to grow and form colonies in culture dishes with and without selective agent (6-TG) for determination of mutations and viability.
Experiment 1:
- In experiment 1, a 5-h treatment with metabolic activation (in the presence of S9-mix) and a 5-h treatment without metabolic activation (in the absence of S9-mix) were performed.
- For the 5-h treatment in the presence and absence of S9-mix, the following concentrations were examined: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL.
- In experiment 1 no insolubility was detected in the final treatment medium at the end of the treatment in the experiment with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
- In the presence of S9-mix (5-h treatment), marked cytotoxicity of the test item was observed at higher concentrations (200 μg/mL concentration showed a relative survival of 15%, 175 μg/mL concentration showed a relative survival of 32% on the survival plates). No cells survived the expression period in the samples of 250 μg/mL concentration. An evaluation was made using data of seven concentrations (concentration range of 200-15.625 μg/mL). 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 evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.
- In the absence of S9-mix (5-h treatment), marked cytotoxicity of the test item was observed at 175 μg/mL concentration (relative survival of 39% on the survival plates). No cells survived the expression period in the samples of 250 μg/mL and 200 μg/mL concentrations. Thus, evaluation was made using data of six concentrations (concentration range of 175-15.625μg/mL). 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 evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

Experiment 2
- Based on the observed results of the mutagenicity plates in experiment 2 with and without metabolic activation, the positive control groups did not fulfil the acceptance criteria, therefore this experiment is considered to be invalid. Furthermore, in some cases unexpected higher colony counts were observed in the test substance treated groups.
- An additional experiment (experiment 3) was performed to provide sufficient data in order to have a valid and conclusive study.

Experiment 3
- In experiment 3, 5-h treatment with metabolic activation (in the presence of S9-mix) and 24-h treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-h treatment in the presence of S9-mix, the following concentrations were examined: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL, and for 24-h treatment in the absence of S9-mix, the following concentrations were examined: 175, 160, 150, 140, 125, 62.5, 31.25 and 15.625 μg/mL.
- In experiment 3 no insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
- In experiment 3, in the presence of S9-mix (5-h treatment), excessive cytotoxicity of the test item was observed at 200 μg/mL concentration (relative survival of 2% on the survival plates) or marked cytotoxicity was observed at 175 μg/mL - concentration (relative survival of 10% on the survival plates), 150 μg/mL concentration (relative survival of 25% on the survival plates). No cells survived the expression period in the samples of 250 μg/mL concentration. Thus, evaluation was made using data of six concentrations (concentration range of 175-15.625 μg/mL) in experiment 3.
Statistically significant increase (at p<0.01 level) was observed at 175 and 125 μg/mL concentration, although the observed values were within the general historical control range. Furthermore, the observed mutant frequency values (18.3 x 10-6 at 175 μg/mL; 11.9 x 10-6 at 125 μg/mL) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6), furthermore the observed increase was not detected in the experiment 1. Therefore, it was concluded as a biologically non-relevant increase. There was no evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.
- In experiment 3, in the absence of S9-mix (24-h treatment), excessive cytotoxicity of the test item (relative survival <10%) was observed at 175-140 μg/mL concentrations and marked cytotoxicity was observed at 125 μg/mL concentration, which was in the appropriate cytotoxicity range (relative survival of 12% on the survival plates). Thus, an evaluation was made using data of four concentrations. 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 evidence of dose response to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.

All the observed mutation frequency values were comparable with the general historical control range and in line with the OECD guideline*. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between treated samples and negative (vehicle) controls.
*Note: The spontaneous mutant frequency is generally between 5 and 20 x10-6 based on the guideline.

For details on results refer to the tables 1-6 under 'any other information on results incl. tables.

VALIDITY OF THE MUTATION ASSAYS
- The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays* (see Tables 5 and 6), and the observed values were in the expected range (5-20 x 10-6) as shown in the OECD No. 476 guideline.
*Note: The mutation frequency values of the negative (vehicle) control (DMSO) in the experiment 1 without metabolic activation and the mutation frequency values of the untreated control in the experiment 3 with metabolic activation, furthermore the negative (vehicle) control (DMSO) in the experiment 3 without metabolic activation were below the lower limit of the historical control range with or without metabolic activation (5-h treatment and 24-h treatment), however all controls were in harmony with the general historical control range. This fact had no impact on the results or integrity of the study, since the results were clearly negative.

- 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 (See Tables 1-4).
- The tested concentration range in the study was considered to be adequate as the defined acceptance criteria of the relevant OECD regarding cytotoxicity produced by the highest evaluated concentrations (approximately 80-90 % toxicity, i.e. approximately 10-20 % relative survival*) were considered to be fulfilled.
*Note: In Assay 1, in case of the experiment without metabolic activation, the relative survival value of the highest surviving concentration of 175 μg/mL was 39%. Although this data is higher than the recommended range, the cells of the closely spaced higher concentration samples (250 and 200 μg/mL) did not survive the expression period. Therefore, this concentration was selected as highest evaluated concentration, and this fact was considered to be acceptable.
- At least four test item concentrations were evaluated in duplicate in each experiment.
- The overall study was considered valid.

Remarks on result:

other: Not mutagenic

Table 1: Survival results of experiment 1

S9 mix	Treatment period (hours)	Studyphase	Test item or control concentration	Totalnumber of colonies	CloningEfficiency(CE)	Relative Survival (%) on plates
+	5	A1	250 µg/mL	ND	ND	ND
			200 µg/mL	176	0.147	15
			175 µg/mL	374	0.312	32
			150 µg/mL	605	0.504	52
			125 µg/mL	725	0.604	63
			62.5 µg/mL	969	0.808	84
			31.25 µg/mL	1115	0.929	96
			15.625 µg/mL	1099	0.916	95
			Negative control (1% (v/v) DMSO)	1158	0.965	100
			Untreated control	1232	1.027	106
			Positive control (DMBA)	55	0.046	5
-	5	A1	250 µg/mL	ND	ND	ND
			200 µg/mL	ND	ND	ND
			175 µg/mL	499	0.416	39
			150 µg/mL	1109	0.924	87
			125 µg/mL	1259	1.049	98
			62.5 µg/mL	1118	0.932	87
			31.25 µg/mL	1237	1.031	97
			15.625 µg/mL	1192	0.993	93
			Negative control (1% (v/v) DMSO)	1279	1.066	100
			Untreated control	1275	1.063	100
			Positive control (EMS)	1001	0.834	78

A1 = experiment 1; + = in the presenceof S9-mix; - = in the absenceof S9-mix                            

DMBA = 7,12-Dimethylbenz[a]anthracene, 15µg/mL; EMS=Ethylmethanesulfonate,0.4µL/mL Negative (vehicle) control =DMSO; DMSO = Dimethyl sulfoxide; ND = No data (No cells were plated for colony growing due to the observed cytotoxicity during treatment.)

Table 2: Survival results of experiment 3:

S9 mix	Treatment period (hours)	Studyphase	Test item or control concentration	Totalnumber of colonies	CloningEfficiency(CE)	Relative Survival (%) on plates
+	5	A3	250 µg/mL	ND	ND	ND
			200 µg/mL	27	0.023	2
			175 µg/mL	116	0.097	10
			150 µg/mL	285	0.238	25
			125 µg/mL	729	0.608	65
			62.5 µg/mL	1036	0.863	92
			31.25 µg/mL	107	0.839	90
			15.625 µg/mL	1065	0.888	95
			Negative control (1% (v/v) DMSO)	1125	0.938	100
			Untreated control	1245	1.038	111
			Positive control (DMBA)	39	0.033	3
-	24	A3	175 µg/mL	11	0.009	1
			160 µg/mL	77	0.064	6
			150 µg/mL	76	0.063	6
			140 µg/mL	106	0.088	9
			125 µg/mL	142	0.118	12
			62.5 µg/mL	1213	1.011	101
			31.25 µg/mL	1321	1.101	110
			15.625 µg/mL	1157	0.964	96
			Negative control (1% (v/v) DMSO)	1204	1.003	100
			Untreated control	1231	1.026	102
			Positive control (EMS)	609	0.508	51

A3 = Assay 3; + = in the presenceofS9-mix; - = in the absenceofS9-mix; DMBA = 7,12-Dimethylbenz[a]anthracene, 15µg/mL; EMS=Ethylmethanesulfonate,0.4µL/mL Negative (vehicle) control =DMSO

DMSO = Dimethyl sulfoxide; ND = No data (No cells were plated for colony growing due to the observed cytotoxicity during treatment.)

Table 3: Viability results of experiment 1

S9 mix	Treatment period (hours)	Studyphase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)
+	5	A1	250 µg/mL	ND	ND
			200 µg/mL	1017	0.848
			175 µg/mL	1137	0.948
			150 µg/mL	1049	0.874
			125 µg/mL	1016	0.847
			62.5 µg/mL	1029	0.858
			31.25 µg/mL	1029	0.858
			15.625 µg/mL	1032	0.860
			Negative control (1% (v/v) DMSO)	1023	0.853
			Untreated control	1084	0.903
			Positive control (DMBA)	966	0.805
-	5	A1	250 µg/mL	ND	ND
			200 µg/mL	ND	ND
			175 µg/mL	1169	0.974
			150 µg/mL	1102	0.918
			125 µg/mL	1070	0.892
			62.5 µg/mL	1154	0.962
			31.25 µg/mL	1063	0.886
			15.625 µg/mL	1072	0.893
			Negative control (1% (v/v) DMSO)	1100	0.917
			Untreated control	1086	0.905
			Positive control (EMS)	854	0.712

A1 = experiment 1; + = in the presenceof S9-mix; - = in the absenceof S9-mix; DMBA = 7,12-Dimethylbenz[a]anthracene, 15µg/mL; EMS=Ethylmethanesulfonate,0.4µL/mL Negative (vehicle) control =DMSO

DMSO = Dimethyl sulfoxide; ND = No data (No cells were plated for colony growing due to the observed cytotoxicity during treatment.)

Table 4: Viability results of experiment 3:

S9 mix	Treatment period (hours)	Studyphase	Test item or control concentration	Total number of colonies	Cloning Efficiency (CE)
+	5	A3	250 µg/mL	ND	ND
			200 µg/mL	ND	ND
			175 µg/mL	1000	0.833
			150 µg/mL	977	0.814
			125 µg/mL	1000	0.833
			62.5 µg/mL	965	0.804
			31.25 µg/mL	1009	0.841
			15.625 µg/mL	988	0.823
			Negative control (1% (v/v) DMSO)	1088	0.907
			Untreated control	1099	0.916
			Positive control (DMBA)	995	0.829
-	24	A3	175 µg/mL	1086	0.905
			160 µg/mL	1006	0.838
			150 µg/mL	670	0.558
			140 µg/mL	695	0.579
			125 µg/mL	836	0.697
			62.5 µg/mL	1186	0.988
			31.25 µg/mL	1009	0.841
			15.625 µg/mL	991	0.826
			Negative control (1% (v/v) DMSO)	1043	0.869
			Untreated control	1127	0.939
			Positive control (EMS)	346	0.288

A3 = Assay 3

+ = in the presenceofS9-mix                             DMBA = 7,12-Dimethylbenz[a]anthracene, 15µg/mL

- = in the absenceofS9-mix                               EMS=Ethylmethanesulfonate,0.4µL/mL Negative (vehicle) control =DMSO

DMSO = Dimethyl sulfoxide

 

ND = No data (No cells were plated for colony growing due to the observed cytotoxicity during treatment.)

Table 5: Mutagenicity results of experiment 1

S9 mix	Treatment period (hours)	Studyphase	Test item or control concentration	Total number of colonies	Mutant frequency
+	5	A1	250 µg/mL	ND	ND
			200 µg/mL	30	8.9
			175 µg/mL	31	8.1
			150 µg/mL	38	11.0
			125 µg/mL	34	10.0
			62.5 µg/mL	28	8.2
			31.25 µg/mL	33	9.7
			15.625 µg/mL	22	6.3
			Negative control (1% (v/v) DMSO)	32	8.8
			Untreated control	30	8.9
			Positive control (DMBA)	1933	600.8**
-	5	A1	250 µg/mL	ND	ND
			200 µg/mL	ND	ND
			175 µg/mL	23	5.9
			150 µg/mL	30	8.2
			125 µg/mL	16	4.5
			62.5 µg/mL	24	6.3
			31.25 µg/mL	26	7.5
			15.625 µg/mL	18	5.0
			Negative control (1% (v/v) DMSO)	21	5.7
			Untreated control	24	6.6
			Positive control (EMS)	1311	466.5**

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control A1 = experiment 1

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

- = in the absenceof S9-mix                               EMS=Ethylmethanesulfonate,0.4µL/mL Negative (vehicle) control =DMSO

DMSO = Dimethyl sulfoxide;

Mutant frequencies refer to 106clonable cells.

ND = No data (No cells were plated for colony growing due to the observed cytotoxicity during treatment.)

Table 6: Mutagenicity results of experiment 3

S9 mix	Treatment period (hours)	Studyphase	Test item or control concentration	Total number of colonies	Mutant frequency
+	5	A3	250 µg/mL	ND	ND
			200 µg/mL	NE	NE
			175 µg/mL	61	18.3**
			150 µg/mL	22	6.7
			125 µg/mL	40	11.9**
			62.5 µg/mL	17	5.3
			31.25 µg/mL	21	6.3
			15.625 µg/mL	28	8.5
			Negative control (1% (v/v) DMSO)	21	5.8
			Untreated control	16	4.4
			Positive control (DMBA)	1639	494.7**
-	24	A3	175 µg/mL	NE	NE
			160 µg/mL	NE	NE
			150 µg/mL	NE	NE
			140 µg/mL	NE	NE
			125 µg/mL	13	4.7
			62.5 µg/mL	16	4.1
			31.25 µg/mL	11	3.3
			15.625 µg/mL	15	4.6
			Negative control (1% (v/v) DMSO)	20	5.8
			Untreated control	18	4.8
			Positive control (EMS)	619	542.3**

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control In the statistical analysis, negative trends were not considered significant.

A3 = Experiment 3; + = in the presenceofS9-mix; - = in the absenceof S9-mix                     

DMBA = 7,12-Dimethylbenz[a]anthracene, 15µg/mL; EMS=Ethylmethanesulfonate,0.4µL/mL Negative (vehicle) control =DMSO; DMSO = Dimethyl sulfoxide; ND = No data (No cells were plated for colony growing due to the observed cytotoxicity during treatment.); NE = Not evaluated (due to the observed cytotoxicity); Mutant frequencies refer to 106clonable cells.

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic to Chinese hamster ovary cells in vitro HPRT assay, both with and without metabolic activation

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance PBBA in CHO K1 Chinese hamster ovary cells at the hprt locus, according to OECD Guideline 476, in compliance with GLP. Treatments were carried out for 5 h with and without metabolic activation (±S9-mix) and for 24 h without metabolic activation (-S9-mix). Dimethyl sulfoxide (1% (v/v)) was used as the vehicle (solvent) of the test substance in this study. Following treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test: Experiment I: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL (5 h treatment in the presence of S9 mix); 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL (5 h treatment in the absence of S9 mix). Experiment III: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL (5 h treatment in the presence of S9 mix); 175, 160, 150, 140, 125, 62.5, 31.25 and 15.625 μg/mL (24-h treatment in the absence of S9-mix). Treatment with the test substance did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies either with or without metabolic activation in experiment I and II. The positive controls gave the anticipated increases in mutation frequency over the controls and the cloning efficiencies and spontaneous mutation frequencies for the negative (vehicle) controls were within the target range. Therefore, the HPRT assay was considered valid. Under the study conditions of thein vitroHPRT assay, the test substance was considered to be non-mutagenic to Chinese hamster ovary cells, both with and without metabolic activation (Varga-Kanizsai, 2019).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitroAmes test:

Anin vitrostudy was conducted to determine the mutagenic potential of the test substance PBBA, according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains ofSalmonella typhimurium(TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA in the Ames plate incorporation and pre-incubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment I was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels and an expanded dose range were selected in Experiment 2 in order to achieve both non-toxic dose levels and the toxic limit of the test substance. Results for the positive and negative controls were in line with the historical laboratory data. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation in both the experiment. Cytotoxicity was observed at 5000 µg/plate with and without metabolic activation for all strain. Under the study conditions of thein vitroAmes test, the test substance was considered to be non-mutagenic toS. typhimuriumstrains, both with and without metabolic activation (Thompson, 2013).

In vitromicronucleus assay:

Anin vitrostudy was conducted to determine the clastogenic and aneugenic potential of the test substance PBBA, according to OECD Guideline 487, in compliance with GLP. The dose levels used in the main experiments were selected using data from the preliminary toxicity test. Three independent exposures were performed. Experiment I used a 4 h exposures in the presence and absence of a standard metabolizing system (2% S9 mix). The following concentrations were evaluated: 45, 90, 180, 240, 300, 360 µg/mL. Experiment II, used a 24 h exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of experiment I. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B. Examined concentrations in this experiment were as follows: 22.5, 45, 90, 120, 180, 240, 300 µg/mL. The metabolic activation system has shown to be functional as results for the positive and negative control were as expected. No biologically relevant increase in the number of binucleated cells containing micronuclei was observed in both experiments (with and without metabolic activation), using a dose range that included approximately 50% reduction in CBPI or greater. Under the study conditions of thein vitromicronucleus assay, the test substance was considered to be non-clastogenic and non-aneugenic to human lymphocytes, both with and without metabolic activation (Morris, 2014).

In vitroHPRT assay: 

Anin vitrostudy was conducted to determine the mutagenic potential of the test substance PBBA in CHO K1 Chinese hamster ovary cells at the hprt locus, according to OECD Guideline 476, in compliance with GLP. Treatments were carried out for 5 h with and without metabolic activation (±S9-mix) and for 24 h without metabolic activation (-S9-mix). Dimethyl sulfoxide (1% (v/v)) was used as the vehicle (solvent) of the test substance in this study. Following treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test: Experiment I: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL (5 h treatment in the presence of S9 mix); 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL (5 h treatment in the absence of S9 mix). Experiment III: 250, 200, 175, 150, 125, 62.5, 31.25 and 15.625 μg/mL (5 h treatment in the presence of S9 mix); 175, 160, 150, 140, 125, 62.5, 31.25 and 15.625 μg/mL (24-h treatment in the absence of S9-mix). Treatment with the test substance did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies either with or without metabolic activation in experiment I and II. The positive controls gave the anticipated increases in mutation frequency over the controls and the cloning efficiencies and spontaneous mutation frequencies for the negative (vehicle) controls were within the target range. Therefore, the HPRT assay was considered valid. Under the study conditions of thein vitroHPRT assay, the test substance was considered to be non-mutagenic to Chinese hamster ovary cells, both with and without metabolic activation (Varga-Kanizsai, 2019).

Justification for classification or non-classification

Based on the results of the in vitro Ames, micronucleus and HPRT assays, the test substance is not required to be classified for genotoxicity according to EU CLP (EC 1272/2008) criteria.