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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Mutagenic activity of N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide was investigated in one bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A), in an in vitro gene mutation study in mammalian cells (mouse lymphoma L5178Y cells) and in two in vitro chromosome aberration study in human lymphocytes and Chinese hamster lung fibroblasts (CHL/IU). Negative results were obtained in all tests with and without metabolic activation.
Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Complete culture medium: RPMI 1640 supplemented with 10% horse serum, 100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Treatment medium: RPMI 1640 supplemented with 5-7.5% horse serum, 100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Selctive medium: RPMI 1640 supplemented with 20% horse serum, 100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I
with and without S9 mix: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10.0 mM
Experiment II
with S9 mix: 0.3, 0.6, 1.2, 2.4, 3.0, 7.0, 8.0, 9.5 and 10.0 mM
without S9 mix: 0.2, 0.5, 1.0, 2.5, 4.0, 5.0, 6.0 and 7.0 mM
Vehicle / solvent:
cell culture medium (RPMI 1640 + 5% horse serum)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
For a short term exposure experiment 1x10(exp7) were suspended in 11 mL RPMI medium with 5% horse serum and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation.
For a long term exposure experiment 5x10(exp6) were suspended in 25 mL RPMI medium with 7.5% horse serum and exposed to designated concentrations of the test item in the absence or presence of metabolic activation. After 24 h the test item was removed by centrifugation.

DURATION
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: experiments with 8 different dose levels

DETERMINATION OF CYTOTOXICITY
- Method: (relative) suspension growth; (relative) clonin efficiency

Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10(exp6) cells
- A dose-dependent increase in mutant is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (>/= 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpreatation of results. Statisitcal methods might be used as an aid in evaluation the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend is negative.
Statistics:
non-parametric Mann-Whitney test
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in the experiments.

No growth inhibition was noted in the pre experiment and in experiment I with and without metabolic activation and in experiment II with metabolic activation. Growth inhibition was observed in pre experiment II and in experiment II without metabolic activation.
In experiment I with metabolic activation the relative total growth (RTG) was 141.2% for the highest concentration (10mM) evaluated. The highest concentration evaluated without metabolic activation was 10 mM with a RTG of 78.7%. In experiment II with metabolic activation the relative total growth (RTG) was 115.7% for the highest concentration (10 mM) evaluated. The highest concentration evaluated without metabolic activation was 7 mM with a RTG of 16.3%.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF) was not exceeded by the induced mutant frequency at any concentration.
No dose-response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the xperimental conditions (with and without metabolic activation).

EMS, MMs and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Addtionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In the described mutagenicity test (MLA) under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The test item N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide was assessed for its potential to induce mutations at the mouse lymphoma kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data fro the pre experiments. In experiment I 10 mM (with and without metabolic activation) was selected as the highest concentrations. In experiments II 10 mM (with metabolic activation) and 7 mM (without metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short term exposure assay. Experiment II without metabolic activation was perfomed as a 24 h long term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

with and without metabolic activation: 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 9.0 and 10.0 mM

Experiment II

with metabolic activation: 0.3, 0.6, 1.2, 2.4, 3.0, 7.0, 8.0, 9.5 and 10.0 mM

and without metabolic activation: 0.2, 0.5, 1.0, 2.5, 4.0, 5.0, 6.0 and 7.0 mM.

No precipitation of the test item was observed in the experiments.

No growth inhibition was noted in the pre experiment and in experiment I with and without metabolic activation and in experiment II with metabolic activation. Growth inhibition was observed in pre experiment II and in experiment II without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 141.2% for the highest concentration (10mM) evaluated. The highest concentration evaluated without metabolic activation was 10 mM with a RTG of 78.7%. In experiment II with metabolic activation the relative total growth (RTG) was 115.7% for the highest concentration (10 mM) evaluated. The highest concentration evaluated without metabolic activation was 7 mM with a RTG of 16.3%.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the xperimental conditions (with and without metabolic activation).

EMS, MMs and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Addtionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide also yielded negative results in an in vitro gene mutation study in mammalian cells in concentration up to 10 mM with and without metabolic activation.

N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide was assessed for its potential to induce chromosome aberrations in human lymphocytes and Chinese hamster lung fibroblasts (CHL/IU) in vitro. The test item did not induce chromosome aberrations. Therefore, N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide is considered to be non-mutagenic in these in vitro chromosome aberration tests when tested up to 10 mM.

In conclusion,N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide is not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study in mouse lymphoma L5178Y cells and the in vitro chromosome aberration studies in the presence and absence of metabolic activation up to the tested concentrations

Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalian cells and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 476 and GLP.

Justification for classification or non-classification

N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide does not have to be not classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assay (up to 10 mM) or in the in vitro chromosome aberration studies in concentrations up to 10 mM.