N-butyl-2,2,6,6-tetramethylpiperidin-4-amine, oligomeric reaction products with N,N'-bis(3-aminopropyl)ethylenediamine and 2,4,6-trichloro-1,3,5-triazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: other: point mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl-2,2,6,6-tetramethylpiperidin-4-amine, oligomeric reaction products with N,N'-bis(3-aminopropyl)ethylenediamine and 2,4,6-trichloro-1,3,5-triazine

EC Number:

500-311-6

EC Name:

N-butyl-2,2,6,6-tetramethylpiperidin-4-amine, oligomeric reaction products with N,N'-bis(3-aminopropyl)ethylenediamine and 2,4,6-trichloro-1,3,5-triazine

Cas Number:

120498-03-5

IUPAC Name:

1,3-Propanediamine,N-N’’-1,2-ethanediylbis- reaction product with 1,3,5-triazine-2,4-diamine,N,N’-dibutyl-6-chloro-N,N’-bis(2,2,6,6-tetramethyl-4-piperidinyl), polymer with N-butyl-4,6-dichloro-N-(2,2,6,6-tetramethyl-4-piperidinyl)-1,3,5-triazin-2-amine

Details on test material:

Name of test material (as cited in study report): TABANOL 4

Method

Target gene:

HGRPT- locus of cultured chinese hamster ovary cells

Test concentrations with justification for top dose:

The final concentrations of the test substance in the culture medium were: 10, 20, 30, 40, 50, and 60µg/ml.
Without metabolic activation: The fourth HGRPT assay the final concentrations of the test substance in teh culture medium were: 5, 10, 20, 25, 30, 35 and 40µg/ml
With metabolic activation: The third HGRPT assay the final concentrations of the test substance were: 2.5, 5, 5, 10, 20, 30, 40, 50 and 60µg/ml.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

The CHO cells were grown in Ham's F-12 culture medium (with Glutamax I), supplemented with heat-inactivated foetal calf serum (10% v/v), and gentamycin (50mg/l) growth medium. The cellss were rountinely cultured in a humidified incubator at 37°C in air containing 5% carbon dioxide. Prior to use in this assay the the CHO cells were generated from stock culture by seeding then in sterile, screw-capped tissue culture flasks containing 10ml Ham's F-12 growth medium. Near-confluent CHO cells were harvested by trypsinization from a number of 75cm² tissue culture flasks, suspended in growth medium and the number of cells were counted. Fo rth eHGRPT-assay, aliquots of 1.5-1.6 x 10^6 CHO cells were added to a number of 75cm² tissue culture flasks containing 10 ml of growth medium. The cells were incubated for approx. 20 hours, in a humidified incubator at 37°C in air containing 5% CO². On teh day following seeding the cells were exposed to the test substance both in teh absence and teh presence of a metabolic activation system.

Evaluation criteria:

The following criteria were used to evaluate the data obtained in the HGRPT assay (Li et al. 1987):
a) the survival (abolute cloning efficiency) of the negative controls should not be less than 50%
b) the mean mutant frequency of th enegative controls should fall within teh range of 0 to 15 6-TG resistant mutants per 1,000,000 clonable cells
c) the mean mutant frequency of positive controls should be higher than 20 6-TG resistant mutants per 1,000,000 clonable cells, and should at least be 5 times higher that teh corresponding negative control
d0 the highest test substance concentration should, if possible, result in a clear cytotoxic response (eg 10-30% of teh relative initial survival). Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% toxicity is considered to be an artefact and not indicative of genetoxicity.
Genetoxicity of the test substance was evaluated using the following criteria:
a) a concentraion-related increase in mutant frequency
b) a reproducible positive response for at least one of the test substance concentrations(eg the mean mutant frequency shoul dbe more than 20 mutants per 1,000,000 clonable cells.

Statistics:

Exact statistical analysis is difficult because the distribution of the number of mutant colonies depends on assumptions of homogenous variance, normal distribution, and the complex processes of cell growth and cell death after treatment with a test substance or a positive control (Li et al. 1987). Therefore evaluation of the data obtained with the HGRPT assay was made using the above described criteria rather than by statistical anlysis.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both the presence and the absence of the S-9 mix the test substance was not toxic to the CHO cells at any of the tested concentrations as apparant from the mean absolute and relative initial cloning efficiencies, which were ≥63.7% in all instances. Some indication of toxicity was apparent from a decrease in mean relative initial yield, though, most markedly in the absence of the S-9 mix.
In teh absence of the S-9 mix, a reproducible positive response of the test substance (>20 mutants per 1,000,000 clonable cells) was found at none of the test substance concentrations tested. The mutant frequencies of the negative controls were in the range generally found (0-15 mutants per 1,000,000 clonable cells), except in one instance were a value of 16.3 was obtained; this value falls in the "grey" area between the range for negative controls and the values for a positive response (>20 mutants per 1,000,000 clonable cells). However, because the other negative control culture (medium control) and all other values for DMSO-controls were in teh negative control range this value is considered a spurious results.
In the presence of the S-9 mix, a reproducible positive response of the test substance was found at none of the test concentrations tested, except for the concentration of 40µg/ml. However, since this increase was to just above the critical value of 20 mutants per 1,000,000 clonable cells, and this response is not dose related, and there was no increase in teh mutant frequency at the next higher dose levels (50 and 60µg/ml) this finding is considered not biologically relevant. The mutant frequencies of the negative controls were in the range generally found (0-15 mutants per 1,000,000 clonable cells).
The positive control substances, EMS (in the absence of S-9 mix) and DMBA (in the presence of S-9 mix), showed expected increases in mutant frequency.

Remarks on result:

other: strain/cell type: HGPRT

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions employed in this examination the test substance does not induce mutation at the HGPRT locus of the CHO cells.