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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19-nov-2009 to 02-feb-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Hexahydro-1,3,5-Trimethyl-S-Triazine- Substance type: Clear pale yellow liquid- Physical state: Liquid- Stability under test conditions: Stable- Storage condition of test material: At room temperature in the dark

Method

Species / strain
Species / strain / cell type:
lymphocytes: human peripheral
Details on mammalian cell type (if applicable):
- Type and identity of media: Blood samplesBlood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.Culture mediumCulture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.Lymphocyte culturesWhole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test: Without and with S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 1292 µg/mLWithout S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 1292 µg/mLCytogenetic test:Without and with S9-mix, 3hr exposure; 24 hr fixation: 3, 12 and 18 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: Test substance is stable in DMSO for at least 4 hours at room temperature under normal laboratory light conditions is demonstrated over the concentration range 0.336 to 134 mg/ml based on neat Tryzine
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/ml for a 3 h exposure period
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Preincubation period: 48 hr- Exposure duration: 3 hr (with and without S9-mix)- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrSPINDLE INHIBITOR (cytogenetic assays): colchicineSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: duplicates in two independent experimentsNUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per cultureDETERMINATION OF CYTOTOXICITY - Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cellsOTHER EXAMINATIONS: - Determination of polyploidy: yes- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human periphal blood
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No- Effects of osmolality: No - Precipitation: No precipitation in the exposure medium was observed up to including the highest tested dose level of 1292 µg/mLRANGE-FINDING/SCREENING STUDIES: - Toxicity was observed at dose levels of 33 µg/mL and above in the absence and presence of S9, at all treatment periodsCOMPARISON WITH HISTORICAL CONTROL DATA: - The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.ADDITIONAL INFORMATION ON CYTOTOXICITY:- Appropriate toxicity was reached at the dose levels selected for scoring.

Any other information on results incl. tables

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

No effects of Hexahydro-1,3,5-Trimethyl-S-Triazine on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

 

It was noted that Hexahydro-1,3,5-Trimethyl-S-Triazine increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner. This may indicate that Hexahydro-1,3,5-Trimethyl-S-Triazine has the potential to disturb mitotic processes and cell cycle progression.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: positiveIn the absence of S9-mix, Hexahydro-1,3,5-Trimethyl-S-Triazine induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. In the presence of S9-mix, Hexahydro-1,3,5-Trimethyl-S-Triazine induced a statistically significant, increase in the number of cells with chromosome aberrations at the highest tested cytotoxic concentration, both when gaps were included and excluded.Finally, it is concluded that this test is valid and that Hexahydro-1,3,5-Trimethyl-S-Triazine is clastogenic in human lymphocytes under the experimental conditions described in this report. Hexahydro-1,3,5-Trimethyl-S-Triazine may have the potential to disturb mitotic processes and cell cycle progression.