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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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:
August 31, 2012 - January 03, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine
EC Number:
425-020-0
EC Name:
Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine
Cas Number:
191680-81-6
Molecular formula:
C50H77N11O2-C168H230N32O8
IUPAC Name:
N2-(2-{[4,6-bis({butyl[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]amino})-1,3,5-triazin-2-yl](3-{[4,6-bis({butyl[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]amino})-1,3,5-triazin-2-yl]amino}propyl)amino}ethyl)-N2-(3-{[4,6-bis({butyl[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]amino})-1,3,5-triazin-2-yl]amino}propyl)-N4,N6-dibutyl-N4,N6-bis[1-(cyclohexyloxy)-2,2,6,6-tetramethylpiperidin-4-yl]-1,3,5-triazine-2,4,6-triamine
Details on test material:
- Physical state: Solid, beige
- Storage condition of test material: At room temperature

Method

Target gene:
hprt (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneus mutant frequency: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I:
4 hours, with and without S9 mix: 0.7, 2.0, 6.0, 18.0, 54.0, (162.0) µg/ml
Experiment II:
24 hours, without S9 mix: (0.7), 2.0, 6.0, 18.0, 54.0, 162.0 µg/ml
4 hours, with S9 mix: 0.7, 2.0, 6.0, 18.0, 54.0, (162.0) µg/ml
numbers in parantheses: these cultures were discontinued.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
without S9: EMS, 0.15 mg/ml; with S9: DMBA, 1.1 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 - 20 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation, visible to the unaided eye, was noted in experiment I at 18.0 μg/mL and above with and without metabolic activation. In experiment II precipitation occurred at 54.0 μg/mL and above without and at 18.0 μg/mL and above with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. An isolated reduction of the cloning efficiency noted at 1155 μg/mL following 4 hours treatment without metabolic activation was judged as precipitation artefact as there was no dose dependent cytotoxicity. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 18.0 μg/mL and above with and without metabolic activation after 4 and 24 hours treatment. In the pre-experiment there was no relevant shift of osmolarity and pH values of the medium even at the maximum concentration of the test item. Based on precipitation of the test item noted in the pre-experiment, the concentration range of the main experiments was selected.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I and II up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results

concentration (µg/ml) P S9 Mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control (DMSO) - 100.0 100.0 100.0 15.0 1.0 100.0 100.0 100.0 18.6 1.0
positive control (EMS) 150.0 - 81.8 103.1 85.3 144.7 9.6 83.7 140.4 105.6 67.6 3.6
test item 0.7 - 98.5 110.7 98.0 18.9 1.3 105.0 134.2 101.2 9.9 0.5
test item 2.0 - 85.1 100.2 93.6 15.5 1.0 95.7 126.3 103.3 14.1 0.8
test item 6.0 - 86.0 94.5 98.7 31.9 2.1 94.4 134.0 103.2 11.2 0.6
test item 18.0 P - 89.1 166.1 106.4 13.4 0.9 102.8 137.8 100.5 14.9 0.8
test item 54.0 P - 77.1 108.7 93.0 14.8 1.0 70.3 132.1 102.3 13.9 0.7
test item 162.0 P - 36.6 culture was not continued# 45.5 culture was not continued#
solvent control (DMSO) + 100.0 100.0 100.0 13.6 1.0 100.0 100.0 100.0 9.7 1.0
positive control (DMBA) 1.1 + 52.1 109.8 88.2 764.5 56.1 53.9 112.0 83.6 647.7 66.7
test item 0.7 + 103.7 92.6 97.6 16.1 1.2 95.0 92.4 92.2 18.7 1.9
test item 2.0 + 98.9 90.0 107.3 10.2 0.7 98.9 108.8 98.2 31.1 3.2
test item 6.0 + 96.3 100.2 87.4 13.4 1.0 100.0 113.1 91.6 18.2 1.9
test item 18.0 P + 107.8 100.5 97.0 12.3 0.9 99.7 115.0 90.9 16.8 1.7
test item 54.0 P + 89.4 77.2 92.8 9.7 0.7 99.2 105.7 88.2 19.3 2.0
test item 162.0 P + 98.3 culture was not continued# 90.1 culture was not continued#
Experiment II / 24h treatment
solvent control (DMSO) - 100.0 100.0 100.0 12.3 1.0 100.0 100.0 100.0 8.9 1.0
positive control (EMS) 150.0 - 92.8 79.9 89.0 394.0 31.9 96.9 249.6 76.8 222.8 25.0
test item 0.7 - 98.9 culture was not continued## 96.4 culture was not continued##
test item 2.0 - 99.2 79.8 83.3 10.9 0.9 97.2 252.6 74.7 23.3 2.6
test item 6.0 - 99.4 99.6 101.8 17.3 1.4 91.3 214.1 77.1 14.0 1.6
test item 18.0 - 86.8 59.7 94.4 15.1 1.2 79.7 192.5 85.6 12.0 1.3
test item 54.0 P - 82.5 87.1 101.9 6.6 0.5 75.8 231.8 87.0 17.6 2.0
test item 162.0 P - 58.6 46.5 98.5 19.4 1.6 60.4 144.2 81.6 13.2 1.5
Experiment II / 4h treatment
solvent control (DMSO) + 100.0 100.0 100.0 18.8 1.0 100.0 100.0 100.0 12.0 1.0
positive control (DMBA) 1.1 + 56.8 83.7 82.6 418.8 22.2 55.4 49.0 77.6 554.9 46.3
test item 0.7 + 97.7 87.3 78.5 11.1 0.6 99.8 92.6 100.7 11.6 1.0
test item 2.0 + 97.9 96.1 107.4 15.1 0.8 98.0 78.0 100.0 9.2 0.8
test item 6.0 + 96.5 97.5 89.0 12.9 0.7 99.8 82.1 89.4 13.0 1.1
test item 18.0 P + 98.2 102.7 88.5 18.3 1.0 96.3 104.0 95.9 14.8 1.2
test item 54.0 P + 95.2 73.9 83.3 12.0 0.6 94.8 66.3 93.0 9.5 0.8
test item 162.0 P + 94.4 culture was not continued# 85.2 culture was not continued#

P Precipitation

# culture was not continued to avoid analysis of too many precipitating concentrations

## culture was not continued as a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 2310 μg/mL used in the pre-experiment was limited by the solubility properties of the test item. The concentration range of the main experiments (0.7 – 162 µg/ml) was limited by precipitation of the test item. The test item was dissolved in THF. Precipitation, visible to the unaided eye, was noted in experiment I at 18.0 μg/mL and above with and without metabolic activation. In experiment II precipitation occurred at 54.0 μg/mL and above without and at 18.0 μg/mL and above with metabolic activation.

No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I and II up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of the first experiment at 2.0 μg/mL with metabolic activation. The effect however, was based on a rather low solvent control of 9.7 mutant colonies/106 cells and has no biological relevance as it was neither dose dependent as indicated by the lacking statistical significance nor reproduced in the parallel culture. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.9 up to 18.8 mutants per 106 cells; the range of the groups treated with the test item was from 6.6 up to 31.9 mutants per 106 cells. EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.