Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-14 to 2015-01-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

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:
yes
Remarks:
no confirmatory (independent repeat) assay after negative results of the mutagenicity assay
Qualifier:
according to guideline
Guideline:
other: Ames (1983) Revised Methods for the Salmonella Mutagenicity Test
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-((2-chlorophenyl)-N-methylcarbonimidoyl)cyclopentan-1-ol
EC Number:
641-894-8
Cas Number:
6740-87-0
Molecular formula:
C13H16ClNO
IUPAC Name:
1-((2-chlorophenyl)-N-methylcarbonimidoyl)cyclopentan-1-ol
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I14LD5177
- Purity: 96.9% (per Analysis Sheet)
- Receipt Date: 22 December 2014
- Expiration date of the lot/batch: 2016-12-31

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) or Tryptophan locus (E. coli strain)
Species / strain
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:
Mutagenicity assay: 10, 25, 50, 75, 150, 300, 600, 1200, 2500 and 5000 μg/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: A solubility test was conducted to determine the vehicle. The 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 these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as 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:
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:
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
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), 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.


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) or Tryptophan (E. coli)

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 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 neither positive nor equivocal.
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate and upwards
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:
at 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:
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 metabolic activation. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.