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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione
EC Number:
248-597-9
EC Name:
1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione
Cas Number:
27676-62-6
IUPAC Name:
1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-1,3,5-triazinane-2,4,6-trione
Constituent 2
Reference substance name:
1,3,5-tris(3,5-di-tert-butyl-4- hydroxybenzyl)-1,3,5-triazine- 2,4,6(1H,3H,5H)-trione
IUPAC Name:
1,3,5-tris(3,5-di-tert-butyl-4- hydroxybenzyl)-1,3,5-triazine- 2,4,6(1H,3H,5H)-trione
Details on test material:
- Physical state: solid
- Analytical purity: 98.2%

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Mixture F-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Concentration range in cytotoxicity/genotoxicity test: 0.22 - 28.0 µg/ml
Concentration range in chromosome analysis test: 7.0 - 28.0 µg/ml
Vehicle / solvent:
Solvent used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without microsomal activation

Migrated to IUCLID6: 0.2 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with microsomal activation

Migrated to IUCLID6: 40.0 µg/ml
Details on test system and experimental conditions:
The cytotoxicity test was performed as an integral part of the mutagenicity test. A series of Falcon flasks was seeded with Chinese hamster ovary cells. The preincubation time before treatment was 29 hours. The substance in DMSO was added (1:100) to the cells in culture medium. In the experiments in which the substance was metabolically activated, 1.0 ml of an activation mixture was added to 9.0 ml of culture medium.

In all four experiments, the cells were exposed to eight concentrations of the test substance ranging from 0.22 to 28.0 µg/ml.
Experiment 1: 7.0 - 28.0 µg/ml, test without activation, duration of treatment: 18 hours, recovery: 0 hours
Experiment 2: 7.0 - 28.0 µg/ml, test with activation, duration of treatment: 3 hours, recovery: 15 hours
Experiment 3: 7.0 - 28.0 µg/ml, test without activation, duration of treatment: 42 hours, recovery: 0 hours
Experiment 4: 7.0 - 28.0 µg/ml, test with activation, duration of treatment: 3 hours, recovery: 39 hours

Two hours prior to harvesting, the cultures were treated with Colcemide 0.4 µg/ml. The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation (methanol:acetic acid, 3:1). Drop preparations were made by the air-drying technique.

The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration . The deteirmination of the mitotic coefficient was performed for all four experiments separately.

One hundred metaphase figures with 19 to 21 chromosomes from cultures of two falcon flasks in the vehicle control, in the treated groups and at least fifty metaphases in the positive controls were examined.





Evaluation criteria:
Criteria for a positive response:
- A test substance is considered to be active in this test system if in comparison to the negative control a marked increase in the number of specific chromosomal aberrations appears or if an increased number of exchange figures appears together with a high number of other specific chromosomal aberrations such as breaks and fragments.
- A concentration-related response in the number of aberrations should be demonstrable.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced mitotic index in all experiments at the highest concentration (28 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The number of chromosome aberrations was within the historical control range at all doses assessed.
The highest concentration of 28.0 µg/ml available for analysis in the first experiment caused 21.1% suppression of mitotic activity. The highest concentration of 28.0 µg/ml available for analysis in the second experiment caused 12.1% suppression of mitotic activity. In the third experiment with a 42 hours treatment period the concentration of 28.0 µg/ml available for analysis caused 29% suppression of mitotic activity. In the fourth experiment (3 hours treatment / 39 hours recovery) the highest concentration of 28.0 µg/ml available for analysis caused 19% suppression of mitotic activity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

EXPERIMENTAL RESULT

percent of metaphases with aberrations
Exposure Time Sampling Time Dose (µg/ml) S9-Mix number of metaphases specific unspecific
18 h 18 h Vehicle - 100 1 2
18 h 18 h 7 - 100 1 2
18 h 18 h 14 - 100 1 2
18 h 18 h 28 - 100 0 3
18 h 18 h Mito-C; 0.2 - 100 50 18
3 h 18 h Vehicle + 100 0 1
3 h 18 h 7 + 100 2 2
3 h 18 h 14 + 100 1 0
3 h 18 h 28 + 100 1 2
3 h 18 h CPA; 40 + 100 36 18
42 h 42 h Vehicle - 100 1 5
42 h 42 h 7 - 100 0 2
42 h 42 h 14 - 100 1 2
42 h 42 h 28 - 100 0 1
3 h 42 h Vehicle + 100 3 1
3 h 42 h 7 + 100 1 0
3 h 42 h 14 + 100 3 2
3 h 42 h 28 + 100 1 5

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with the test substance.
Executive summary:

The clastogenic activity of the test substance was assessed in experiments without metabolic activation at concentrations between 7.0 and 28.0 µg/ml with 18 hours incubation time and with 42 hours incubation time. In the experiments with a metabolic activating system the concentrations between 7.0 and 28.0 µg/ml with 3 hours incubation followed by 15 hours recovery period and with 3 hours incubation followed by 39 hours recovery period were assessed. One hundred metaphases were examined from the vehicle control and from the cultures treated with the various concentrations of the test substance. At least fifty metaphases each from the appropriate positive controls were analyzed. Neither with nor without metabolic activation a significant increase in the number of chromosome aberrations were observed. The number of chromosome aberrations was within the historical control range at all doses assessed. It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with the test substance.