Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
White to off-white powder.
Storage Conditions: At room temperature.
Specific details on test material used for the study:
Identification: Maropitant base (CJ-11,972)
Batch (Lot) Number: 334115
Expiry date: 14 April 2020 (retest date)
Physical Description: White to off-white powder
Purity/Composition: 99.9%
Storage Conditions: At room temperature
Additional information
Test Facility test item number: 210137/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Stable
Solubility in vehicle: Dimethyl sulfoxide: Not indicated
Stability in vehicle: Dimethyl sulfoxide: Not indicated

Method

Target gene:
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
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:
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 is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene,
which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and
2.5 µg/plate, respectively.
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose-range finding test with the
strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest
concentration of Maropitant base (CJ-11,972) used in the subsequent mutation assays was
5000 µg/plate or the level at which the test item inhibited bacterial growth.
The tested concentrations were: 0.54, 1.7, 5.4, 17, 52, 164, 500 µg/plate
Vehicle / solvent:
Dimethyl sulfoxide (DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: TA 1537 without S9 2.5µg/plate; 2-Aminoanthracene, all strains with S9 1-15µg/plate
Details on test system and experimental conditions:
At least five different doses (increasing with approximately half-log steps) of the test item
were tested in triplicate in each strain. The above mentioned dose-range finding study with
the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation
experiment. In the second part of this experiment, the test item was tested both in the absence
and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a
follow-up experiment with additional parameters, the test item was tested both in the absence
and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each
strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(10^9 cells/mL) of one of the tester strains, 0.1 to 0.2 mL of a dilution of the test item in DMSO
and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in
case of non-activation assays). The ingredients were mixed on a Vortex and the content of
the top agar tube was poured onto a selective agar plate. After solidification of the top agar,
the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this
period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria
and tryptophan independent (Trp+) for Escherichia coli) were counted.

The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates
with sufficient test item precipitate to interfere with automated colony counting were counted
manually. 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.
Evaluation criteria:
See below
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

See attachment

Applicant's summary and conclusion

Conclusions:
Maropitant base (CJ11,972) 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 Maropitant base (CJ-11,972) 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 study procedures described in this report were based on the most recent OECD and EC guidelines. Batch 334115 of the test item was a white to off-white powder with a purity of 99.9%. The vehicle of the test item was dimethyl sulfoxide. In the dose-range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the top dose level of 5000 μg/plate. 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 at dose levels of 164 μg/plate and upwards and in tester strain WP2uvrA at dose levels of 1600 μg/plate and upwards. Results of this dose-range finding test were reported as part of the first mutation assay. Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at the concentration ranges of 0.54 to 164 µg/plate in tester strain TA1537 (absence and presence of 5% (v/v) S9-mix)) and tester strain TA98 (absence of S9-mix) and at the concentration ranges of 0.54 to 500 µg/plate in the tester strains TA1535 (absence and presence of S9-mix) and TA98 (presence of S9-mix). The test item did not precipitate on the plates at this 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. In a follow-up experiment of the assay with additional parameters, the test item was tested up to concentrations of 500 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 µg/plate in tester strainWP2uvrA in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at the dose levels of 1500 and 1000 µg/plate and upwards in the absence and presence of S9-mix, respectively. 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. Maropitant base (CJ-11,972) 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Maropitant base (CJ11,972) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.