Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-01-15 to 2015-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-03-02 to 2017-03-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16KB4361
- Purity: 99.5%
- Description: Clear colorless liquid
- Receipt Date: 2017-02-07
- Expiration date of the lot/batch: 2017-05-27

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2 to 8°C, protected from light and under inert gas (Argon)

Target gene:
Histidine locus (S. typhimurium strains); Tryptophan locus (E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Initial Toxicity-Mutation Assay: 10.0, 25.0, 50.0, 75.0, 150, 300, 600, 1200, 2500 and 5000 μg/plate with and without S9-mix;
Confirmatory Mutagenicity Assay: 10, 25, 50, 75, 150, 300, 600, 1200, 2500 and 5000 µg/plate with and without S9-mix;

Since the test item was soluble at 50 mg/mL (= 5000 µg/plate, the maximum recommended concentration according to the Guideline), 5000 µg/plate was selected as the top dose of the initial toxicity-mutation assay.
In the mutation assay, the top dose was selected based on the toxicity observed in the initial toxicity-mutation assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on information provided by the Sponsor. In a study previously conducted at BioReliance (AE13YE.502005ICH), the test article formed a clear solution in DMSO at approximately 50 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9: 1.0 μg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9: 1.0 μg/plate for TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9: 1.0 μg/plate for WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9: 75 μg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: 1.0 μg/plate for TA98, TA1535 ; 2.0 μg/plate for TA100, TA1537 ; 15 μg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test article and the vehicle, all test article dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.
One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 100 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test article aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium strains) or tryptophan (E. coli strain)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn

Rationale for test conditions:
Solubility limitations: since the test item was soluble in DMSO at 50 mg/mL (= the highest recommended dose level by the OECD 471 Guidance), the maximum dose tested in the mutagenicity assay was 50 mg/mL (= 5000 μg/plate).
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative if it is neither positive nor equivocal.
Statistics:
no data
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 300 µg/plate or higher, and upwards (without S9); at 600 µg/plate or higher, and upwards (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction in revertant counts at 5000 µg/plate with S9 in the Confirmatory Assay only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not specified
- Precipitation:
Initial toxicity-mutation assay: Precipitate was observed beginning at 300 μg per plate during dosing. No precipitate was observed during background lawn evaluation/scoring.
Confirmatory mutation assay: Precipitate was observed beginning at 300 μg per plate during dosing. No precipitate was observed during scoring.

RANGE-FINDING/SCREENING STUDIES:
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and ten dose levels (10 to 5000 µg/plate) of the test article, in triplicate, in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : All criteria for a valid study were met as described in the protocol.
- Positive historical control data: The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. This validity criterium was met.
- Negative (solvent/vehicle) historical control data: Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls. This validity criterium was met.
Conclusions:
Interpretation of results: negative with and without metabolic activation
The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9, when stored according to the recommended conditions (2 to 8 °C under Argon).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Ames (1983) Revised Methods for the Salmonella Mutagenicity Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-chlorophenyl cyclopentyl ketone
EC Number:
229-802-0
EC Name:
2-chlorophenyl cyclopentyl ketone
Cas Number:
6740-85-8
Molecular formula:
C12H13ClO
IUPAC Name:
2-chlorophenyl cyclopentyl ketone
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): JNJ-54582034-AAA (T003642)
- Physical state: liquid
- Appearance: yellow liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020709377
- Purity: 96.1% (per Analysis Sheet)
- Description: Clear colorless liquid
- Receipt Date: 2014-12-22
- Expiration date of the lot/batch: 2016-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:
histidine locus (S. typhimurium strains); tryptophan locus (E. coli strains)
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
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Mutagenicity assay: 10, 25, 50, 75, 150, 300, 600, 1200, 2500 and 5000 μg per plate.

A range-finding assay was not required. The test item was observed to be soluble in DMSO at 50 mg/mL (=5000 μg/plate). The maximum dose tested in the mutagenicity assay was 5000 μg per plate, the maximum dose recommended by the OECD 471 Guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was selected as the vehicle of choice in consultation with the Sponsor, and based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in DMSO at approximately 50 mg/mL, the maximum final concentration.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9: 75 μg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
2-nitrofluorene
Remarks:
Without S9: 1.0 μg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9: 1.0 μg/plate for TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9: 1.0 μg/plate for WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: 1.0 µg/plate for TA98 and TA1535, 2.0 µg/plate for TA100 and TA1537, 15 µg/plate for WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of sterile water for each 100 mL of minimal top agar. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).
To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test article and the vehicle, all test article dose levels and the vehicle used in the mutagenicity assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.
One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 100 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains) or tryptophan (E. coli strains)

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneuos with exposure)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn

Rationale for test conditions:
Solubility limitations: since the test item was soluble in DMSO at 50 mg/mL (= the highest recommend ed dose level by the OECD 471 Guidance), the maximum dose tested in the mutagenicity assay was 50 mg/mL (= 5000 μg/plate).
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated as positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value.
- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was either positive nor equivocal.
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
up to 3.1- and 10.0-fold increases in the presence and absence of S9 activation, respectively
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 600, 1200 or 2500 μg/plate and upwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: A solubility test was conducted using sterile water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL. Based on the solubility results, DMSO was selected as vehicle.
- Precipitation: No precipitate was observed.

RANGE-FINDING/SCREENING STUDIES: A range-finding assay was not required

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): All criteria for a valid study were met as described in the protocol.
- Positive historical control data: The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. This validity criterium was met.
- Negative (solvent/vehicle) historical control data: All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60. This validity criterium was met.

Applicant's summary and conclusion

Conclusions:
The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did cause positive mutagenic responses with tester strain WP2 uvrA in both the presence and absence of metabolic activation, when stored at room temperatue instead of according to the recommended conditions (2 to 8 °C under Argon).
The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.