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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test: not mutagenic up to 1600 µg/plate, with and without metabolic activation (OECD 471, GLP, Key study, rel. 1)

- In vitro micronucleus assay: not clastogenic or aneugenic in human lymphocytes, with and without metabolic activation (OECD489, GLP, key study, rel.1)

- in vitro gene mutation assay (MLA): mutagenic in the presence of metabolic activation (OECD490, GLP, key study, rel.1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 October 2017 - 19 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and acc ording to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
The highest concentration of the test item used in the subsequent mutation assay was the level at which the test item inhibited bacterial growth unless the test item exhibited limited solubility.

Main study:
- Experiment 1 (direct plate assay): TA1535, TA1537 and TA98: Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1600 µg/plate (based on the results of the dose-range finding test)
- Experiment 2 (pre-incubation assay): TA1535, TA1537, TA98, TA100 and WP2uvrA: Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1000 µg/plate (based on the results of the first mutation assay)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Merck, Darmstadt, Germany)
- Justification for choice of solvent/vehicle: Not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate (direct plate assay) and 5 µg/plate (pre-incubation assay) in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9: 2.5 µg/plate (direct plate assay) in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 15 µg/plate (pre-incubation assay) in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate (direct plate assay) and 10 µg/plate (pre-incubation assay) in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate (direct plate assay) and 650 µg/plate (pre-incubation assay) in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate (direct plate assay) and 10 µg/plate (pre-incubation assay) in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: 2.5 µg/plate (direct plate assay) and 2.5 µg/plate (pre-incubation assay) in DMSO for TA1535 and TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: 1 µg/plate (direct plate assay) and 1 µg/plate (pre-incubation assay) in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: 1 µg/plate (direct plate assay) and 5 µg/plate (pre-incubation assay) in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: 15 µg/plate (direct plate assay) and 15 µg/plate (pre-incubation assay) in DMSO for WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Plate incorporation and pre-incubation methods
- Cell density at seeding: 10^9 cell/mL

DURATION :
- Direct plate assay (first experiment), exposure duration: 48+/-4h in dark at 37+/-1°C
- Preincubation assay (second experiement), exposure duration: 48+/-4h in dark at 37+/-1°C

NUMBER OF REPLICATIONS: Doses of test item were tested in triplicate in each strain in the absence and presence of S9-mix. Two independent experiments were conducted: a direct plate assay and a pre-incubation assay.

NUMBER OF CELLS EVALUATED: Not specified

DETERMINATION OF CYTOTOXICITY :
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were
observed.

OTHER EXAMINATIONS:
- Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

- ACCEPTABILITY OF THE ASSAY:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Testing Facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
In the dose-range finding study, the test item precipitated on the plates at dose levels of 1600 μg/plate and upwards.
In the first mutation experiment (direct plate assay), the test item precipitated on the plates at dose levels of 512 μg/plate and upwards (at the end but not at the start of the incubation period).
In the second mutation experiment (pre-incubation assay), the test item precipitated on the plates at the dose levels of 512 μg/plate and upwards (at the end but not at the start of the incubation period).

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and the presence of microcolonies, was observed in tester strain TA100 at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. No mutagenicity was observed up to those doses in both strains. Results of this dose-range finding test were reported as part of the first mutation assay.

HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for WP2uvrA (absence of S9-mix) in the second experiment.
The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 2 times greater than the concurrent solvent control values and clear negative results were obtained, this deviation in the mean plate count of the positive control had no effect on the results of the study.
Positive and negative (solvent/vehicle) historical control data are provided in attachment (see section "attached background material").

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Direct plate assay: Cytotoxicity, as evidenced a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella
typhimurium tester strains in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
- Pre-incubation assay: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all
Salmonella typhimurium tester strains in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
Conclusions:
Under the test conditions, 1,3-Bis (4-hydroxybenzoyl) benzene is not mutagenic in the Salmonella typhimurium (TA1535, TA1537, TA98 and TA100 strains) reverse mutation assay and in the Escherichia coli (WP2uvrA strain) reverse mutation assay, both in the absence and presence of S9-metabolic activation.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471, EU Method B.13/14 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA100) and Escherichia coli (WP2uvrA) were exposed to the test substance, 1,3-Bis (4-hydroxybenzoyl) benzene, dissolved in dimethyl sulfoxide at the following concentrations both in the presence and absence of metabolic activation system (S9 -mix).

Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate

Main study:

- Experiment 1 (direct plate assay): TA1535, TA1537 and TA98: Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1600 µg/plate

- Experiment 2 (pre-incubation assay): TA1535, TA1537, TA98, TA100 and WP2uvrA: Without and with S9-mix: 5.4, 17, 52, 164, 512 and 1000 µg/plate

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and the presence of microcolonies, was observed in tester strain TA100 at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 1000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at the dose levels of 512 μg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella tester strains in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+ ) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+ ) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that 1,3-Bis (4-hydroxybenzoyl) benzene is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-Oct-2018 to 23-May-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MMHBB-002/18
- Expiration date of the lot/batch: 01-APR-2021
- Purity test date: 09-APR-2018
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: Blood from donors aged 26, 27 or 28 were used (sex not reported).
- Whether whole blood or separated lymphocytes were used: whole blood treated with heparin.
- Whether blood from different donors were pooled or not: blood from separate donors were used in each experiment
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
Environmental conditions: humid atmosphere of 80 - 100% (actual range 43 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 33.1 - 37.9°C).
Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate (Trinova Biochem GmbH, Giessen, Germany) prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg)
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies).
The solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.2 mL S9-mix was added to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was
1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported
Test concentrations with justification for top dose:
At a concentration of 625 μg/mL 1,3-Bis (4-hydroxy benzoyl) benzene precipitated in the culture medium after 3 and 24 hours.
A dose-range finding assay was performed to assess cytotoxicity and doses for the main assays.

* Dose range-finding assay: 39, 78, 156, 313, 625 and 1250 μg/ml culture medium, for 3 and 24 hours without S9-mix, or for 3 hours with S-9 mix.
Due to a very steep toxicity after 24 hrs exposure, the dose-range finding test was repeated. The blood cultures were treated with 0.16, 0.63, 2.5, 10 and 40 μg/mL culture medium.

* First cytogenetic assay, with or without S9-mix:
- 10, 40, 80, 100, 120, 140 and 160 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).
Scoring of micronuclei was performed on the following dose levels:
- Without S9-mix: 10, 100 and 140 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).
- With S9-mix: 10, 80 and 160 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).

* Second cytogenetic assay, without S9-mix:
- Without S9-mix: 1, 5, 10, 15, 20, 25 and 30 μg/mL culture medium (24 hours exposure time, 24 hours harvest time).
Scoring of micronuclei was performed on the following dose levels:
- Without S9-mix: 1, 10 and 15 μg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide (DMSO). In the dose-range finding study, the stock solution was treated with ultrasonic waves until the test item had completely dissolved. The highest tested concentration was determined by the solubility of the test item in the culture medium.
- Justification for percentage of solvent in the final culture medium: 1.0% (v/v). Recommended maximum percentage in the test guideline.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL)
Phytohaemagglutinin) were cultured for 46 ± 2 hours.
- Test substance added in medium, in suspension.
- Appropriate vehicle and positive controls were included.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours and 24 hours in the absence of S9-mix, or for 3 hours in the presence of S9-mix (duplicate cultures)
- Harvest time after the end of treatment (sampling/recovery times):
After 3 hours exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle).
The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.

FOR MICRONUCLEUS ASSAY:
- cytokinesis blocked method used for micronucleus assay: Cytochalasine B (5 μg/mL) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.

- Methods of slide preparation and staining technique used including the stain used:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol:acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. The slides were marked with the study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of 500 cells (with a maximum deviation of 5%) per culture, with duplicate cultures.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Three analyzable concentrations are scored for micronuclei.
*At least 1000 (with a maximum deviation of 5%) binucleated cells per culture are examined by light microscopy for micronuclei.
* In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture are scored for micronuclei separately.

The following criteria for scoring of binucleated cells will be used:
* Main nuclei that are separate and of approximately equal size.
* Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
* Main nuclei that are linked by nucleoplasmic bridges.

The following cells will not be scored:
* Trinucleated, quadranucleated, or multinucleated cells.
* Cells where main nuclei are undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
* The diameter of micronuclei should be less than one-third of the main nucleus.
* Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
* Micronuclei should have similar staining as the main nucleus

- Determination of polyploidy: yes, scoring cells with one, two or more nuclei (multinucleated cells).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- Supplementary information relevant to cytotoxicity: Based on the results of the DRF test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.
In case no statistically significant increase is observed in the cultures dosed with the lowest dose of MMC-C and CP, both doses are scored for the presence of micronuclei. The number of examined bi- or mononucleated cells in the positive control groups might be <1000, due to cytotoxicity.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
All slides were randomly coded before examination of micronuclei and scored.
The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). An intermediate dose level was also scored.
Rationale for test conditions:
The dose levels for the 1st assay were selected based on the dose-range finding test. Three dose levels with and without S9-mix were selected for scoring of micronuclei.
The first assay was conducted with a short treatment of 3 hours followed by a recovery period of 27 hours prior to harvest. A second assay was performed to confirm the results of the 1st assay, and modified conditions were applied, with a 24-hour treatment (in the absence of S9-mix).
Evaluation criteria:
ACCEPTABILITY CRITERIA:
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

ANALYSIS OF RESULTS:
- POSITIVE (clastogenic or aneugenic) if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

NEGATIVE (not clastogenic or aneugenic) if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.
Statistics:
Chi-square test, one-sided
Cochran Armitage trend test
Key result
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
lack of dose-related increase in the 1st assay without S9; a positive trend with S9 was within the 95% control limits of the distribution of the vehicle control database. No effect in the confirmatory assay with a longer treatment time without S9.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
test up to the cytotoxic concentration and limit of precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: the pH of a concentration of 625 μg/mL was 8.1, compared to 8.2 in the solvent control.
- Data on osmolality: The osmolarity of a concentration of 625 μg/mL was 384 mOsm/kg compared to 398 mOsm/kg in the solvent control.
- Possibility of evaporation from medium: no data. Unlikely based on the vapour pressure of the test item.
- Water solubility: due to its limited water solubility the test item was dissolved in DMSO as a stock solution prior to treatment of cell cultures
- Precipitation and time of the determination: At a concentration of 625 μg/mL 1,3-Bis (4-hydroxy benzoyl) benzene precipitated in the culture medium after 3 and 24 hours

RANGE-FINDING STUDY:
The highest tested concentration was determined by the solubility of the test item in the culture medium. At a concentration of 625 μg/mL 1,3-Bis (4-hydroxy benzoyl) benzene precipitated in the culture medium after 3 and 24 hours.
Doses: 39, 78, 156, 313, 625 and 1250 μg/ml culture medium
Treatment time: 3 and 24 hours in the absence of S9-mix, and for 3 hours in the presence of S9-mix.
Due to very steep toxicity after 24 hours exposure to the test item, the dose-range finding test was repeated. The blood cultures were treated with 0.16, 0.63, 2.5, 10 and 40 μg/mL culture medium

STUDY RESULTS
- treated cultures: See result tables 1& 2
Table 1: 3 hr-treatment, 27-hr harvest time, with and without S9-mix
In the absence of S9-mix, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei. No significant trend was observed.
In the presence of S9-mix, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei. In addition a significant trend (p=0.014) was observed (Cochran Armitage trend test). However, both in the absence and presence of S9-mix, these increases were within the 95% control limits of the distribution of the historical vehicle control database (Table 3). Therefore these increases were considered not biologically relevant.
No increases were observed in the number of mononucleated cells with micronuclei.

A second assay was conducted with modified conditions, with a 24-hr treatment period in the absence of S9-mix (Table 2): the test item did not induce a statistically significant or biologically
relevant increase in the number of mono- and binucleated cells with micronuclei.

- Concurrent vehicle negative and positive control data : see detailed result Tables 1 & 2.
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of mononucleated cells with micronuclei in the first experiment and produced a statistically significant increase in the number of binucleated cells with micronuclei in the first and second experiment.
The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei in the first and second experiment and produced a statistically significant increase in the number of binucleated cells with micronuclei in the first experiment. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that
the metabolic activation system (S9-mix) functioned properly.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Negative (solvent/vehicle) historical control data: see Table 3
- Positive historical control data: see table 4

Table 1 - First assay : Cytokinesis-block Proliferation Index and scoring of mononucleated and binucleated cells with micronuclei in human lymphocyte cultures treated with 13BHBB

> Without metabolic activation, 3 hours exposure, 27 hours harvest time

     

number of mononucleated cells with micronuclei (1)

       number of binucleated cells with micronuclei (1)
       1000 1000   2000  1000  1000  2000
 conc. (µg/ml)  mean CBPI  % cytostasis  A  B  A+B  A  B  A+B
 0 1.33 0 0  0  0  2  2  4
 10 1.36  -9 0  1  1  2  3  5
 40  1.30  10  -  -  -  -  -  -
 80  1.20 41  -  -  -  -  -  -
 100  1.22  34 0  2  2  7  5  12*
120  1.19  41  -  -  -  -  -  -
 140  1.16  52 1  0  1  2  2  4
 160  1.10  69  -  -  -  -  -  -
0.25 MMC-C  1.23  31 3  0  3*  20  15  35***
0.38 MMC-C  1.16  50  -  -  -  -  -  -

 0.1 Colch.

 1.13

 62

15

 18

 33***

 20

 26

 46***

- : not scored

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

(1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

> With metabolic activation, 3 hours exposure, 27 hours harvest time

     

      number of mononucleated cells with micronuclei (1)

        number of binucleated cells with micronuclei (1)

 

 

 

 1000

 1000

 2000

 1000

 1000

 2000

  conc. (µg/ml)

 mean CBPI

 % cytostasis

 A

B

A+B

A

 B

A+B

0

 1.98

0

 0

 0

 0

 2

 0

 2

 10

 1.90

8

 0

 0

 0

 3

 2

 5

 40

 1.69

29

 -

 -

 -

 -

 -

 80

 1.62

36

 0

 0

 0

 3

 3

 6

 100

 1.60

39

 -

 -

 -

 -

 -

 -

 120

 1.58

40

 -

 -

 -

 -

 -

 -

 140

 1.56

42

 -

 -

 -

 -

 -

 -

 160

 1.38

61

 0

 1

 1

 7

 4

 11**

 15 CP

 1.33

67

 3

 3

 6**

 28

 25

 53***

 17.5 CP

 1.28

72

 -

 -

 -

 -

 -

 -

- : not scored

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

(1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

Table 2 - Second Assay: Cytokinesis-block Proliferation Index and scoring of mononucleated and binucleated cells with micronuclei in human lymphocyte cultures treated with 13BHBB

> Without metabolic activation, 24 hours exposure time, 24 hours harvest time

     

     number of mononucleated cells with micronuclei (1)

     number of binucleated cells with micronuclei (1)

 

 

 

 1000

 1000

 2000

 1000

 1000

 2000

 conc. (µg/ml)

 mean CBPI

% cytostasis 

 A

 A+B

 A

B

 A+B

 0

 1.29

 0

 0

 1

1

 3

 0

 3

 1

 1.28

 3

 2

 0

2

 2

 2

 4

 5

 1.25

 13

 -

 -

 -

 -

 -

 -

 10

 1.20

 33

 2

 2

 4

 1

 2

3

 15

 1.15

 50

 1

 0

 1

 1

 2

3

 20

 1.06

 79

 -

 -

 -

 -

 -

 -

 25

 1.04

 87

 -

 -

 -

 -

 -

 -

 30

 1.03

 90

 -

 -

 -

 -

 -

 -

 0.15 MMC-C

 1.15

 50

 1

 2

 3

 21

 16

 37***

 0.23 MMC-C

 1.11

 61

 -

 -

 -

 -

 -

 -

 0.05 Colch.

 1.00

 100

 13

 14

 27***

 0 (2)

 1 (2)

 1

- : not scored

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

(1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

(2) 71 and 98 binucleated cells were scored for the presence of micronuclei, respectively

Table 3 - Historical Negative Control Data for the solvent control

   Mononucleated        Binucleated      
   + S9-mix  - S9-mix     + S9-mix  - S9-mix   
   3-hr exposure  3-hr exposure  24-hr exposure  3-hr exposure  3-hr exposure  24-hr exposure

 mean number of micronucleated cells

(per 1000 cells)

 0.98  0.92  0.88  3.46  3.46  3.35
 SD  1.36  0.95  1.06  2.30  2.31  2.48
 n  162  164  154  162  164  154

 Upper control limit

(95% control limits)

 3.67  3.22  3.52  8.62  8.50  9.23

 Lower control limit

(95% control limits)

 -1.71  -1.38  -1.77  -1.69  -1.57  -2.53

SD: Standard Deviation

n = Number of observations

Distribution of historical negative control data from experiments performed bewteen September 2015 and September 2018

Table 4 - Historical Positive Control Data

   Mononucleated     Binucleated      
   - S9-mix     + S9-mix  - S9-mix   

 

 3-hr exposure  24-hr exposure  3-hr exposure  3-hr exposure  24-hr exposure

 Mean number of micronucleated cells

(per 1000 cells)

 27.22

 32.03

 18.48

 24.93

 22.77

SD

 12.95

 17.29

 8.68

 9.85

 11.94

 n

 163

 152

 178

 174

 176

 Upper control limit

(95% control limits)

 42.92

 64.63

 34.88

 45.27

 47.60

  Lower control limit

(95% control limits)

 -8.28

 -0.56

 2.07

 4.58

 -2.06

SD: Standard Deviation

n = Number of observations

Distribution of historical positive control data from experiments performed bewteen September 2015 and September 2018

Conclusions:
Under the test conditions, in a valid test, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in human lymphocytes exposed for a 3-hour treatment time, with or without metabolic activation, and for a 24-hour treatment time without metabolic activation.
The test item is not clastogenic or aneugenic in human lymphocytes.
Executive summary:

1,3-Bis (4-hydroxy benzoyl) benzene was assessed for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments that were conducted in compliance with the most recent OECD guideline 487.

The test item, a white flake powder was dissolved in dimethyl sulfoxide following a solubility test. Appropriate dose levels were selected based on the results of a dose-range finding assay.

In the first cytogenetic assay, the test item was tested up to 140 and 160 μg/mL for a 3 hours exposure time with a 27 hours harvest time, in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, the test item was tested up to 15 μg/mL for a 24 hours exposure time with a 24 hours harvest time, in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

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

In the first cytogenetic assay in the absence of S9-mix, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei. No significant trend was observed. In the presence of S9-mix, the test item also induced a statistically significant increase in the number of binucleated cells with micronuclei. In addition a significant trend (p=0.014) was observed. However, both in the absence and presence of S9-mix, these increases were within the 95% control limits of the distribution of the historical vehicle control database. Therefore these increases were considered not biologically relevant. No increases were observed in the number of mononucleated cells with micronuclei.

In the second cytogenetic assay with prolonged treatment time, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and bi-nucleated cells with micronuclei.

In conclusion, this test is valid and the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29-Apr-2019 To 12-Aug-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
The experiment without S9 did not meet acceptance criteria but it was no repeated because the experiment with S9 showed positive result. In the mutation experiment with S9, one solvent control was not used.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from the American Type Culture Collection, (ATCC, Manassas, USA) (2001)
- Suitability of cells: Recommended test system in international guidelines
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: verified
- Cell cycle length, doubling time or proliferation index : no data
- Modal number of chromosomes: not specified
- Periodically checked for karyotype stability: no data
- Periodically ‘cleansed’ of spontaneous mutants: yes, cleansing was performed prior to the DRF and mutagenicity tests

MEDIA USED
- Type and composition of media:
* basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2mM L-glutamin
* Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
* Exposure medium for 3-hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
* Exposure medium for 24-hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
* Selective medium: basic medium, supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/mL trifluorothymidine (TFT).
* Non-selective medium: basic medium, supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
- CO2 concentration: 5.0 ± 0.5% CO2
- humidity level, temperature: 80 - 100% humidity, 37.0 ± 1.0°C.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate purchased from Trinova Biochem GmbH, Giessen, Germany, prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix : per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The above solution is filtered (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction is added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : 4% (v/v) in the exposure medium
- quality controls of S9 : metabolic capability was confirmed in the experiment with the positive control cyclophosphamide
Test concentrations with justification for top dose:
The test item was assessed in a solubility assay, and was found to precipitate in the exposure medium at concentrations of 500 µg/ml and 125 µg/ml and above in the 3-hr and 24-hr treatment, respectively. Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 500 μg/mL and 125 µg/mL exposure medium in the 3 hour and 24 hour treatment, respectively.
A dose-range finding assay was used to select the dose ranges for the main tests.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: based on solubility test (visual homogeneity).

- Justification for percentage of solvent in the final culture medium: maximum recommended concentration in the test guideline (1% v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration : a single was used per concentration ;
For the mutation frequency, 5 96-well plates were used per concentration, each well containing 2000 cells in selective medium (TFT-selection medium). For the cloning efficiency, one cell was added per well, using 2 96-well plates/concentration.
- Number of independent experiments : a repeat independent experiment was not performed following the positive results found in the 1st experiment with S9.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium. 8x10E6 cells per culture were used (10E6 cells/ml) for the 3-hr treatment with and without S9.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hrs with and without S9;
The experiment with a 24-hr exposure without S9 was not necessary as positive results were obtained with the 3-hr treatment with S9.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): cells subcultured for 2 days after the treatment period
- Selection time (if incubation with a selective agent): 11-12 days in 96-well microtiter plates in selective medium with trifluorothymidine (TFT)
- Method used: 96- microwell plates.
- If a selective agent is used (trifluorothymidine): 5 µg/ml TFT, 11-12 days incubation period
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: for mutation frequency, 4x10E6 cells were subcultured every day.
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: For the mutation assays: cloning efficiency and relative total growth (RTG). For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a
96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.

- Any supplementary information relevant to cytotoxicity: the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
METHODS FOR MEASUREMENTS OF GENOTOXICITY
Mutation frequency (MF):
A total number of 9.6 x 10E5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection)
The plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the MF were scored with the naked eye or with the microscope.
The colonies were divided into small and large colonies.
Evaluation criteria:
ACCEPTABILITY CRITERIA
a) Absolute cloning efficiency of the solvent controls (CEday2) between 65 and 120% to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) spontaneous MF in the solvent control ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) Suspension growth (SG) over the 2-day expression period for the solvent controls between 8 and 32 for the 3-hr treatment, and between 32 and 180 for the 24-hr treatment.
d) positive control should demonstrate an absolute increase in the total MF, i.e, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10E-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10E-6 above the concurrent solvent control (a small colony IMF of 150 x 10-6).
If (one of) the acceptability criteria are not met, the test will be rejected and repeated. In case no clear conclusion for positive or negative result can be made an additional confirmation study will be performed to confirm the study results.

ANALYSIS
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data (HCD) range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the microwell version, the GEF is 126 x 10E-6.

* positive/mutagenic: if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. Any observed increase should be biologically relevant and will be compared with the HCD range.
* equivocal/questionable: if no clear conclusion for positive or negative result can be made after an additional confirmation study.
* negative/not mutagenic): if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
Usually not used in MLA assay.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RSG of 28% at 60 µg/ml, 4% at 70 µg/ml
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
However, no increase in MF seen in the experiment rejected due to solvent control CE out of the preset range
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 30 µg/ml (RSG: 49% at 30µg/ml, 3% at 40 µg/ml)
Vehicle controls validity:
not valid
Remarks:
Both solvent controls were found below the CE acceptance criteria of 65-120%. One solvent control with a minor deviation (64%) was used for calculations; MF and SG were within the required criteria.
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not measured because precipitation was observed
- Data on osmolality: not measured because precipitation was observed
- Possibility of evaporation from medium: not likely
- Precipitation and time of the determination: precipitation at 500 µg/ml in the 3-hr treatment, and at 125 µg/ml in the 24-hr treatment
- Definition of acceptable cells for analysis: survival
- Other confounding effects: none known

RANGE-FINDING/SCREENING STUDIES: results are reported in Tables 1 and 2

STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in Table 3
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. The results showed that the test conditions were adequate and the metabolic activation system (S9-mix) functioned properly.

Criteria for data analysis and interpretation:
- the interpretation of the MLA results was based on a GEF value of 126; MF results of treated were compared to the MF of the corresponding controls + 126.

Genotoxicity results are reported in Table 3
- Results from cytotoxicity measurements: Relative total growth (RTG) and cloning efficiency (CEday2)
- Genotoxicity results:
In conditions without S9, the cloning efficiency of both controls were outside acceptability criteria. Calculations of MF were done using the control that had a cloning efficiency (64%) just below the criteria (65%). The results did not show a trend in increase of mutation frequency up to the test dose of 30 µg/ml that had a RTG of 43%.
In conditions with S9, 2 test concentrations increased the mutation frequency above the 95% control limits of the historical negative control database and also above the GEF + MF(controls) (185 per 10E6 survivors).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Negative (solvent/vehicle) historical control data: in Table 4
- Positive historical control data: in Table 5

Dose-range finding tests

Table 1 - Dose range-finding test: cytotoxicity following 3-hour treatment

 Dose  cell count after 24-h of subculture   cell count after 48-h of subculture  SG (1)  RGS (2)
 (µg/ml)  (cells/ml x 10E5)   (cells/ml x 10E5)    %
  without metabolic activation     
 Solvent control (DMSO)  4.3  6.8  15  100
 31  5.2  5.9  15  105
 63  0.4 (4)  1.4  0  2
 125  0.1 (4)  0.3  0  0
 250  0.1 (4)  0.2  0  0
 500  0.2 (4)  0.5  0  0
  with metabolic activation
 Solvent control (DMSO)  5.7  6.3  18  100
 31  5.0  6.8  17  95
 63  1.8  5.1  5  25
 125  0.3 (4)  0.5  0  0
 250  0.0 (4)  0.1  0  0
 500 (3)  0.1 (4)  0.2  0  0

(1) Suspension growth

(2) Relative Suspension Growth

(3) The test item precipitated in the exposure medium

(4) since less than 1.25 x 10E5 cells/ml were present, no subculture was performed

Table 2 - Repeat Dose range-finding test: cytotoxicity following 3-hour treatment

 Dose  cell count after 24-h of subculture   cell count after 48-h of subculture  SG(1)  RGS(2)
 (µg/ml)  (cells/ml x 10E5)   (cells/ml x 10E5)    %
  without metabolic activation    
 Solvent control (DMSO)  4.5  7.5  17  100
 30

 3.0

 7.2

 11

 64

 60

 1.0 (4)

 4.3

 2

 13

 80

 0.4 (4)

 1.1

 0

 1

 100

 0.2 (4)

 0.2

 0

 0

 125

 1.2 (4)

 5.4

 3

 19

 250

 0.1 (4)

 0.1

 0

 0

500 (3)

 0.1 (4)

0.1

 0

 0

with metabolic activation 

 Solvent control (DMSO)

4.6

 7.3

 17

 100

30

2.7

6.3

 9

 51

60

2.3

6.7

 8

 46

 80

 1.4

 5.1

 4

 21

 100

 0.4 (4)

 0.4

 0

 0

 125

  0.4 (4)

 0.2

 0

 0

 250

  0.1 (4)

 0.1

 0

 0

 500

  0.2 (4)

 0.2

 0

 0

(1) Suspension growth

(2) Relative Suspension Growth

(3) The test item precipitated in the exposure medium

(4) since less than 1.25 x 10E5 cells/ml were present, no subculture was performed

Table 3 - Mutation Experiment 1A

 dose

 RSG

 CE day2

 RCE

 RTG

 mutation frequency per 10E6 survivors     

 (µg/ml)

 (%)

 (%)

 (%)

 (%)

 total

 (small

 large)

 

without metabolic activation

 

3-hour treatment

 SC1

 100

 64

 100

 100

 77

 (35

 40)

 1.3

 87

 52

 88

 77

 88

 (53

 33)

 2.5

 96

 68

 115

 110

 55

 (28

 26)

 5

 94

 58

 98

 92

 92

 (50

 40)

 10

 97

 48

 80

 78

 96

 (47

 47)

 20

 77

 67

 113

 87

 63

 (38

 24)

 30

 49

 52

 88

 43

 62

 (41

 20)

 40

 3

 84

 141

 4

 128

 (60

 61)

 MMS

 86

 43

 72

 62

 514

 (295

 192)

 

with metabolic activation

 

 3-hour treatment  

 SC1

 100

 71

 100

 100

 59

 (36

 22)

 5

 105

 47

 66

 69

 69

 (18

 50)

 10

 103

 59

 83

 86

 71

 (21

 49)

 20

 81

 58

 81

 65

 108

 (50

 54)

 30

 70

 58

 82

 57

 108

 (61

 44)

 40

 48

 63

 89

 43

 156

 (65

 83)

 50

 37

 64

 90

 33

 152

 (64

 80)

 60

 28

 63

 89

 25

 308

 (138

 140)

 70

 4

 76

 106

 4

 420

 (154

 201)

 CP

 49

 27

 38  19  1865  (1143  514)

notes:

- in the absence of metabolic activation the solvent controls were found outside the cloning efficiency criteria; the solvent control with the CE closest to the criteria range was used for calculations.

- in the presence of metabolic activation the solvent control SC2 was found outside the MF criteria, and not used for the mutation frequency determination

- bolded values correspond to MF treated with a GEF= MF(controls) + 126

Table 4 - Historical Negative Control Data of the spontaneous Mutation Frequencies of the solvent control for the Mouse Lymphoma Assay

 

           Mutation frequency per 10E6 survivors

 

 without S9-mix  

 with S9-mix

 

 3-hr exposure

 24-hr exposure

 3-hr exposure

 mean

101

98

100

 SD

30

31

30

 n

279

262

293

 Upper control limit

(95% control limits)

170

162

165

 Lower control limit

(95% control limits)

31

34

36

SD: Standard Deviation

n = Number of observations

Distribution of historical negative control data from experiments performed between September 2015 and September 2018

Table 5 - Historical Control Data of the Mutation Frequencies of the positive controls for the Mouse Lymphoma Assay

             Mutation frequency per 10E6 survivors
   without S9-mix    with S9-mix
   3-hr exposure  24-hr exposure  3-hr exposure

 mean

803 695 1545
 SD 253 223 887
 n 142 132 151

 Upper control limit

(95% control limits)

1533 1270 3954

 Lower control limit

(95% control limits)

72 119 -864

SD: Standard Deviation

n = Number of observations

Distribution of historical negative control data from experiments performed between September 2015 and September 2018

Conclusions:
1,3-Bis (4-hydroxy benzoyl) benzene is mutagenic in the TK mutation test system in the presence of metabolic activation.
Executive summary:

The mutagenic potential of 1,3-Bis (4-hydroxybenzoyl) benzene, a white flaky powder, was assessed by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the presence and absence of S9-mix with a 3 hour treatment period.

The study procedures described in this report were based on the most recent OECD 490 guideline. The vehicle of the test item was dimethyl sulfoxide.

1,3-Bis (4-hydroxy benzoyl) benzene was tested up to concentrations of 40 μg/mL and 70 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 3 and 4% in the absence and presence of S9-mix, respectively.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the presence of S9-mix, 1,3-Bis (4-hydroxy benzoyl) benzene induced increases above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (185 per 106 survivors). Although the relative total growth at the highest tested concentration was too low according to the guideline (severe toxicity was observed), the increase in the mutation frequency at the lower concentration (RTG 25%) was also above the 95% control limits of the distribution of the historical negative control database and was also above the GEF + MF(controls) and therefore considered biologically relevant.

In the absence of S9-mix, 1,3-Bis (4-hydroxy benzoyl) benzene did not induce a biologically relevant increase in the mutation frequency but the cloning efficiency of the solvent controls did not fulfill the acceptability criteria. This part of the experiment was repeated but did not induce appropriate toxicity and was therefore rejected as no dose levels were selected for mutation frequency determination.

Since the assay is clearly positive in the presence of S9-mix, repeating the experiment in the absence of S9-mix would have given no further information and will not change the overall conclusion of the mutagenicity of the test item.

In conclusion, 1,3-Bis (4-hydroxy benzoyl) benzene is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The substance was not tested in vivo as no exposure is expected.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genetic toxicity assessment was based on three reliable in vitro studies.

Reverse mutation assay:

A reverse gene mutation assay was performed according to the OECD test guideline No. 471, EU Method B.13/14 and in compliance with GLP, using the strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA100) and Escherichia coli (WP2uvrA).

Under appropriate conditions of test, no mutagenic effects were observed up to the cytotoxic dose levels, with and without metabolic activation. These results were confirmed in a follow-up experiment.

In vitro Micronucleus assay:

In vitro mouse lymphoma assay:

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the currently available data, no self-classification is proposed for the substance for the hazard class Germ Cell Mutagenicity according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.