Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2017 - 02 January 2018
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Identification: 1,4-Bis (4-hydroxy benzoyl) benzene
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)
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)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain
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 1600 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
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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:
no 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 512 μg/plate and upwards.
In the first mutation experiment (direct plate assay), the test item precipitated at the start and at the end of the incubation period at concentrations of 164 and 512 µg/plate and upwards, respectively.
In the second mutation experiment (pre-incubation assay), the test item precipitated at the start and at the end of the incubation period at the concentration of 512 and 164 µg/plate and upwards, respectively.

RANGE-FINDING/SCREENING STUDIES:
Due to precipitate, the bacterial background lawn could not be determined at dose levels of 5000 µg/plate. At dose levels of 1600 µg/plate and lower, the bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 at the dose level of 5000 μg/plate in the 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.

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.
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 by a decrease in the number of revertants, was observed in the tester strain TA100 in the presence of S9-mix at the dose level of 5000 µg/plate. No toxicity was observed in the absence of S9-mix or in the other tester strains.
- Pre-incubation assay: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Remarks on result:
other: Cytotoxicity was observed in the tester strain TA100 in the presence of S9-mix at the dose level of 5000 µg/plate, only during the first experiment: direct plate assay. No toxicity was observed in the absence of S9-mix or in the other tester strains.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, 1,4-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, 1,4-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 1600 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 512 μg/plate and upwards. Due to precipitate, the bacterial background lawn could not be determined at dose levels of 5000 µg/plate. At dose levels of 1600 µg/plate and lower, the bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 at the dose level of 5000 μg/plate in the 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. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 1600 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 dose levels of 164 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

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 1,4-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.