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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: KIHATSU No 603; Test guidelines for bacterial mutagenic testing, (Notice No 77, 1 September 1988, Ministry of Labour, amended subsequently by Notice No 67, 2 June 1997, and KIHATSU No 413, 2 June 1997)
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): DPMA (Di-propirenglycol-methylether)
- Physical state: Clear, transparent liquid.
- Analytical purity: > 99%
- Impurities (identity and concentrations): not specified in the report
- Composition of test material, percentage of components: not specified in the report
- Isomers composition: 4 isomers
- Purity test date: not specified in the report
- Lot/batch No.: MK 13011TD1
- Expiration date of the lot/batch: not specified in the report
- Stability under test conditions: not specified in the report
- Storage condition of test material: brown bottle stored at ambient

Method

Target gene:
his-/his+
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified in the report
- Periodically "cleansed" against high spontaneous background: not specified in the report
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified in the report
- Periodically "cleansed" against high spontaneous background: not specified in the report
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified in the report
- Periodically "cleansed" against high spontaneous background: not specified in the report
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified in the report
- Periodically "cleansed" against high spontaneous background: not specified in the report
Metabolic activation:
with and without
Metabolic activation system:
S-9 metabolic activation system
Test concentrations with justification for top dose:
Dose range-finding study: 0, 19.5, 78.1, 313, 1250, or 5000 ug/plate
Main study: 0, 313, 635, 1250, 2500, or 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: As DPMA was easily soluble in water, injection water (Lot No. 99J14A) was used as the solvent.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: AF-2, 9-aminoacridine, sodium azide and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 2 per concentration

NUMBER OF CELLS EVALUATED: not specified in the report

DETERMINATION OF CYTOTOXICITY
- Method: number of colonies

Frozen stock cultures of Salmonella typhimurium were transferred to nutrient rich broth and incubated at 37°C until reaching a cell density between 1 and 2E9 cells/ml for each of the four tester strains (TA98, TA100, TA1535, & TA1537).

Results were considered positive if the number of colonies exceeded twice background for any of the strains at any dose and if a dose-response relationship was observed in any strain, with or without S-9 activation. In addition the positive response had to be reproducible in a second experiment. Results were considered negative if the revertant counts did not exceed twice background for any tester strain and the negative response was reproducible in a second experiment

Evaluation criteria:
For a test to be considered valid, if each value of positive and negative control plates remained within the standard range calculated from the background data and there was no abnormal finding in the sterility test.
If the treatment plate did not show any growth inhibition caused at least a doubling of mean revertany colonies over the negative control and the reproducible positive results were observed in dose dependent way, it was concluded as a mutagenic activity.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and Escherichia coli, strain (WP2uvrA)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Not toxic at highest concentration.
Remarks on result:
other: strain/cell type: Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and Escherichia coli, strain (WP2uvrA)
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

DPMA did not induce a mutagenic response in any tester strain at any concentration, with or without S-9 metabolic activation and toxicity did not interfere with the assay and negative and positive controls fell within historical control limits
Executive summary:

Test substance, DPMA was tested for mutagenic potential using histidine dependent autotrophic mutants of Salmonella typhimurium, strains TA 98, TA1537, and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA in two independent mutation tests in the presence (with metabolic activity) and absence (without metabolic activity) of liver preparations (S9-mix) by preincubation method.

DPMA dissolved in the distilled water for injection (Japan Pharmacopoeia) up to 5000 μg/plate were tested, and 6 dose levels were separated by 2 of common ratio in the preliminary study. In the definitive study, 5000μg/plate was a highest dose level and then 5 dose levels were selected by 4 of common ratio as set those in the preliminary test.

No evidence of mutagenic activity to show the increases in the reverse mutation colonies exceeding 2 folds of those of negative control value was seen at any dose level off DPMA in either mutation test. The values calculated for the concurrent negative and positive controls of each strain were within the range of the background data.

Under the conditions of the study, it was concluded that DPMA shows no evidence of mutagenic activity in this bacterial system.