Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jan 2018 to 30 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Based on laboratory historical data these deviations are considered not to affect the study integrity.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(4-bromo-3-formylphenoxy)benzonitrile
EC Number:
822-547-1
Cas Number:
906673-54-9
Molecular formula:
C14H8BrNO2
IUPAC Name:
4-(4-bromo-3-formylphenoxy)benzonitrile
Test material form:
solid: particulate/powder
Details on test material:
Off-white powder
Specific details on test material used for the study:
Purity/Composition: 98.7% Test item storage: At room temperature protected from light Stable under storage conditions until: 20 November 2019 (retest date) (taken from label)

Method

Target gene:
histidine
Species / strain
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 Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
Test concentrations with justification for top dose:
In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay.
In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537 and TA98 in the direct plate assay. The test item was tested beyond a precipitating dose level.
Since too many dose levels showed severe toxicity, an additional experiment was performed to complete the data of the first experiment. In this additional mutation experiment (experiment 1A), the test item was tested up to concentrations of 52 µg/plate in the tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix. The test item did not precipitate on the plates at this dose level.
Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. The test item was tested in tester strain TA100 at the concentration range of 1.7 to 512 µg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA the test item was tested at the concentration range of 17 to 5000 µg/plate in absence and presence of S9-mix.
In the second mutation experiment, the test item was tested up to concentrations of 164 µg/plate in the absence of S9-mix and up to 512 µg/plate in the presence of S9-mix in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. Precipitation of the test item was only observed in the tester strains TA1537 (presence of S9-mix) and TA98 (absence and presence of S9-mix).
The test item was tested in tester strain TA100 up to the dose level of 300 µg/plate in the direct plate assay.
Vehicle / solvent:
dimethyl sulfoxide.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Test System Salmonella typhimurium bacteria and Escherichia coli bacteria Rationale Recommended test system in international guidelines (e.g. OECD, EC). Source Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)

The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was checked to confirm the tryptophanrequirement, UV-sensitivity and the number of spontaneous revertants at least every year. Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Cell Culture Preparation of bacterial cultures Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for a test. Agar plates Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Sigma). Top agar Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C. Environmental conditions All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.1 - 38.1°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD, EC).
Evaluation criteria:
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 Charles River Den Bosch. 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. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA1535, TA1537, TA98 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that PF-06932437 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of PF-06932437 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).   The test was performed in two independent experiments in the absence and presence of  S9-mix, at first a direct plate assay was performed and secondly a pre-incubation assay. Additional experiments were performed to complete the data for the direct plate assay. The study procedures described in this report were based on the most recent OECD and EC guidelines. Groton Batch GR12302 of PF-06932437 was an off-white powder.  A correction factor of 1.013 was used to correct for the purity (98.7%).  The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay.  The test item was tested up to or beyond a precipitating dose level.  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 tester strain TA100 in the absence and presence of S9-mix and in WP2uvrA in the absence of S9-mix.  Since PF-06932437 precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined.  PF-06932437 showed a biologically relevant increase in the number of colonies on the plates in tester strain TA100 in the absence of  S9-mix.  In the first mutation experiment, the test item was tested up to concentrations of  5000 µg/plate in the tester strains TA1535, TA1537 and TA98 in the direct plate assay.  The test item was tested beyond a precipitating dose level.  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.  Since PF-06932437 precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined.  No increase in the number of revertants was observed upon treatment with PF-06932437. Since too many dose levels showed severe toxicity, an additional experiment was performed to complete the data of the first experiment.  In this additional mutation experiment (experiment 1A), the test item was tested up to concentrations of 52 µg/plate in the tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix.  The test item did not precipitate on the plates at this dose level.  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.  No increase in the number of revertants was observed upon treatment with PF-06932437. Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method.  The test item was tested in tester strain TA100 at the concentration range of 1.7 to 512 µg/plate in the absence and presence of  S9-mix.  In tester strain WP2uvrA the test item was tested at the concentration range of 17 to 5000 µg/plate in absence and presence of S9-mix.  

In tester strain WP2uvrA, the test item was tested up to or beyond a precipitating dose level.  In tester strain TA100 the test item did not precipitate on the plates.  Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was only observed in tester strain TA100 in the absence and presence of  S9-mix.  No increase in the number of revertants was observed upon treatment with  PF-06932437. In the second mutation experiment, the test item was tested up to concentrations of  164 µg/plate in the absence of S9-mix and up to 512 µg/plate in the presence of S9-mix in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay.  Precipitation of the test item was only observed in the tester strains TA1537 (presence of S9-mix) and TA98 (absence and presence of S9-mix).  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 three tester strains in the absence and presence of S9-mix.  No increase in the number of revertants was observed upon treatment with PF-06932437. Since in tester strain TA100 in the dose range finding test of the first mutation experiment, the test item induced an up to 2.1-fold increase in the number of revertant colonies in the absence of S9-mix, an additional third experiment was performed to verify this result.  The test item was tested in tester strain TA100 up to the dose level of 300 µg/plate in the direct plate assay. Cytotoxicity was observed at dose levels of 150 µg/plate and above.  No increase in the number of revertants was observed upon treatment with PF-06932437. 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. In the first experiment (direct plate assay), PF-06932437 showed increases in the number of revertant colonies with tester strain TA100, in comparison to the solvent control in the absence and presence of S9-mix.  The increase observed in the absence of S9-mix was above the laboratory historical control data range and was up to 2.1-fold the concurrent vehicle control.  However this increase was not seen in the second experiment (pre-incubation assay) or in the repeat experiment (direct plate assay).  Furthermore, this increase was seen at one toxic dose level only.  The increase observed in the presence of S9-mix was above the laboratory historical control data range and was up to 1.5-fold the concurrent vehicle control. However this increase was not more than two times the concurrent vehicle control and just above the historical control value.  Therefore, these increases are considered to be not biologically relevant and PF-06932437 is considered to be not mutagenic. All other tester strains did not induce a significant dose-related increase in the number of revertant (His+) colonies (TA1535, TA1537 and TA98) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.   In conclusion, based on the results of this study it is concluded that PF-06932437 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.