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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-01 to 2011-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Details described in study specific records
Specific details on test material used for the study:
- Name of test material (as cited in study report): JEFFCAT DPA
- Substance type: Yellowish liquid
- Physical state: Liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: 0.24 wt% water, 8,8 meq/g total amine
- Lot/batch No.: 0H519
- Storage condition of test material: Room temperature, stored protected from light without desiccant

Method

Target gene:
Histidine dependence (S. typhimurium) -> frameshift mutations
Tryptophan locus (E. coli) -> basepair mutations
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate
Confirmatory mutagenicity assay: 50, 150, 500, 1500 and 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Water was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (1 µg/plate for TA98, TA1535 and TA1537), (2.0 µg/plate for TA100) and (15 µg/plate for WP2 uvr A)
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: 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 metabolic activation Migrated to IUCLID6: 1.0 µg/plate for TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: 75 µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation Migrated to IUCLID6: 1,000 µg/plate for WP2 uvrA
Details on test system and experimental conditions:
An initial toxicity-mutation assay and a confirmatory mutagenicity assay was performed

METHOD OF APPLICATION: in agar (plate incorporation)

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

SELECTION AGENT (mutation assays): selective minimal agar

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: A dose level is considered toxic if one or both of the following criteria are met: (1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn.

OTHER: Automated data collection system were used for the collection of data or analysis.
Evaluation criteria:
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.
Data sets for tester strains TA1535 and TA1537 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. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase 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 will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test
- Precipitation: No precipitation was observed.
- Sterility results: Contaminant colonies were observed on the pre-dosing sterility plates for the vehicle and Sham mix in the confirmatory mutagenicity assay. Since no contamination was observed on the assay plates, the Study Directer has concluded that this had no adverse impact on the integrity of the data or the validity of the study conclusions. No contaminant colonies were observed on the remaining sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES: In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg per plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Excessive toxicity, as reductions in revertant counts beginning at 150 µg per plate was observed, therefore tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was spilled by the technician during dosing and there was not enough remaining to dose tester strains Wp2uvrA in the presence of S9 activation. Therefore, tester strain WP2uvrA in the presence of S9 activation was also retested in the retest of confirmatory mutagenicity assay.In the retest experiment, no positive mutagenic responses were observed with tester strain TA1537 in the absence of S9 activation and tester strain WP2 uvrA in the presence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 µg/plate. Neither precipitate nor toxicity was observed.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
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.