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EC number: 203-614-9 | CAS number: 108-77-0
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-02-24 to 2012-06-25
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- 2,4,6-trichloro-1,3,5-triazine
- EC Number:
- 203-614-9
- EC Name:
- 2,4,6-trichloro-1,3,5-triazine
- Cas Number:
- 108-77-0
- Molecular formula:
- C3Cl3N3
- IUPAC Name:
- trichloro-1,3,5-triazine
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
Constituent 1
Method
- Target gene:
- thymidine kinase (TK) locus in L5178Y TK +/- mouse lymphoma cells
Species / strain
- Species / strain / cell type:
- mammalian cell line, other: L5178Y mouse lymphoma cell line
- Details on mammalian cell type (if applicable):
- - Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes (against the TK -/- phenotyp)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat S9 fraction and an energy producing system comprised of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate
- Test concentrations with justification for top dose:
- 0, 12.5, 50, 125, 500, 1250 and 2500 µg/mL (preliminary cytotoxicity experiment)
0,1.56, 3.13, 6.25, 12.5 and 25 µg/mL (mutagenicity test) - Vehicle / solvent:
- Acetone
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Methylmethanesulfonate (without metabolic activation), 3-Methylcholanthrene (with metabolic activation)
- Remarks:
- Positive controls were not used in the cytotoxicity assay
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3h and 24h without S9; 3h with S9 (two independent assays)
- Expression time (cells in growth medium): 2 - 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days
SELECTION AGENT (mutation assays): 5-trifluoro-thymidine
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; - Evaluation criteria:
- Acceptance and evaluation criteria:
- The mutant frequency in the negative control falls within the normal range (historical mean value).
- The plating efficiency (Plating efficiency step 1 and step 2) of the negative control is ≥ 50%.
- At least one concentration of the positive control induces a clear increase in the mutant frequency (the mutant frequency of the positive control is ≥ 2 times the historical mean value of the negative control) with and without S9.
- The mean mutant frequencies (MF) in the negative (vehicle) control cultures fell within the normal range (50 to 170 mutants per 106 viable cells)
- At least one positive control should show either an absolute increase in mean total mutant frequencies (MF) of at least 300 x 10-6 (at least 40% of this should be in the small colony MF), or an increase in small colony mutant frequency of at least 150 x 10-6 above the concurrent vehicle control
- The mean relative Total Growth (RTG) for the positive controls should be greater than 10%
- The mean cloning efficiencies (CE) of the negative controls from the Mutation Experiments were between the range 65% to 120% two days after treatment
- The mean suspension growth (SG) of the negative control replicates from the Mutation Experiments was between the range 8 to 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments
- There should be no excessive heterogeneity between replicate cultures.
Results and discussion
Test results
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 25 µg/mL with and without S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: In a preliminary experiment without and with metabolic activation pronounced to complete cytotoxicity (decreased survival) was noted starting at a concentration of 50 µg/mL. Hence, in the main study the concentration-range of 1.56 to 25 µg/mL was used in the experiments without and with metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative controls were consistent with the historical control data.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation
Under the conditions of the present mouse lymphoma forward mutation assay in vitro no mutagenicity was observed for Cyanuric chloride with and without metabolic activation.
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