A mixture of: 2,2,6,6-tetramethylpiperidin-4-yl-hexadecanoate; 2,2,6,6-tetramethylpiperidin-4-yl-octadecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

This substance was negative in a guideline Ames assay (bacterial reverse mutation assay) with and without metabolic activation.

This substance was negative in a guideline chromosomal aberrations assay in V79 cells, with and without metabolic activation.

This substance was negative in a guideline gene mutation assay in mouse lymphoma L5178Y cells, with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study report obtained through inquiry process. SNIF file obtained from ECHA.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium, other: LT 2, TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Concentration range in the main test (without metabolic activation): 8 to 5000 pg/ plate
Concentration range in the main test (with metabolic activation): 8 to 5000 pg/plate.

Vehicle / solvent:

Solvent: Ethanol

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 1000 pg/plate

Species / strain:

S. typhimurium, other: LT 2, TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 picograms/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: LT 2, TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

40 picograms/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: main test

Conclusions:

Interpretation of results (migrated information):
negative

This substance was negative in a guideline Ames assay (bacterial reverse mutation assay) with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study report obtained through inquiry process. SNIF file obtained from ECHA.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

also according to OECD 473

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cytogenicity (B10)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Vehicle / solvent:

Water

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 5 hours. Exposure period (without metabolic activation): 5 hours. Fixation time : 18 and 24h

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest dose only, 1:25 dilution

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dilution of 1:40, resulting in 50% MI

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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

This substance was negative in a guideline chromosomal aberrations assay in V79 cells, with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-04 - 2014-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP-study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: recommendations of the "International Workshop on Genotoxicity Tests Workgroup" (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- -3.7.2C mouse lymphoma cells;
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for
mycoplasma contamination. Cell density was preferably kept below 1 x 106 cells/mL.

Metabolic activation:

with and without

Metabolic activation system:

(adult male Wistar) rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone

Test concentrations with justification for top dose:

In the first experiment, the test substance was tested up to concentrations of 17 μg/mL and 80 μg/mL in the absence and presence of 4% (v/v) S9-mix, respectively.
In the second experiment, the test substance was tested up to concentrations of 10 μg/mL and 90 μg/ mL in the absence and presence of 8% (v/v) S9-mix.

Vehicle / solvent:

The test substance was dissolved in ethanol.

Untreated negative controls:

yes

Remarks:

see Solvent / vehicle controls

Negative solvent / vehicle controls:

yes

Remarks:

= negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9-mix) Methyl methanesulfonate (MMS) was used as positive c ontrol, with metabolic activation (+S9-mix) Cyclophosphamide (CP) was used.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3h (1st experiment), 24 h (2nd experiment)
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): The microtiter plates for CE(day2) and MF were
incubated for 11 or 12 days.
- Fixation time (start of exposure up to fixation or harvest of cells):
SELECTION AGENT (mutation assays): Selective medium consisted of basic medium supplemented
with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluo
rothymidine (TFT)
STAIN (for cytogenetic assays): After the incubation period, the plates for the TFT-selection were
stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) to each well.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: For determination of the MF a total number of 9.6x10E5 cells/
concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selectiv
e medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a
total number of 9.6x10E5 cells/concentration were plated in ten 96-well microtiter plates, each well
containing 1000 cells in selective medium (TFT-selection).
DETERMINATION OF CYTOTOXICITY
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by
treating 8x10E6 cells (10E6 cells/mL for 3 hours treatment) or 5x10E6 cells (1.25 x 10E5 cells/mL for
24 hours treatment) with a number of test substance concentrations increasing with approximately
half log steps. The cell cultures for the 3 hours treatment were placed in sterile 30 mL centrifuge tu
bes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24
hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test substance w
as tested in the absence and presence of 4% (v/v) S9-fraction. Since the test substance was poorly
soluble in the exposure medium, the highest tested concentration was 333 μg/mL exposure medium.
OTHER: List of protocol deviations
1. During the subculturing period the humidity was recorded to be outside the range of 80 - 100% as
specified for a maximum of 6 hours in the first mutation experiment with a minimum of 63% and for a
maximum of 6.5 hours in the second mutation experiment with a minimum of 53%.
Evaluation: The deviations in the humidity were caused by adjustment of the humidity in the incubator
after opening of the incubator door. The culture flasks were covered with a screw cap (during subcul
turing), all microtiter plates were covered with a lid and wrapped in aluminum foil (determination of
mutant frequency) (continuous oxygen and CO2 exchange enabling cell respiration was ensured)
and normal cell growth was observed in the negative controls, these deviations of the humidity had
no effect on the results of the study.
2. For determination of the CE(day2) a total number of 164 wells was used for the positive control in
the first experiment (presence of S9-mix).
Evaluation: Since 164 out of the 192 wells could be used for the determination of the CE(day2) (de
viation of 15%), this deviation in the number of wells had no effect on the results of the study.
3. In the second experiment, the cloning efficiency of both solvent controls (CE(day2)) (absence
of S9-mix) and of one of the solvent controls (CE(day2)) (presence of S9-mix) were not within the
protocolled range (65 - 120%).
Evaluation: The values of 176 and 127% (absence of S9-mix) and 121% (presence of S9-mix) were
outside the limit of the range (120%). Clear negative results were obtained. Therefore this deviation
in the cloning efficiency had no effect on the results of the study.
The study integrity was not adversely affected by the deviations.
Evaluation criteria
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CE(day2)) is between 65 and 120% in order
to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between
8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10E6 survivors, and for CP not below
700 per 10E6 survivors.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

other: negative control was vehicle control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test substance precipitated in the exposure medium at concentrations of 100 μg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 μg/mL.
RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 μg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 22% at the test substance concentration of 10 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 33 μg/mL and above.
In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 33 g/mL compared to the solvent control. Hardly any cell survival was observed at test substance concentrations of 100 μg/mL and above.
In the absence of S9-mix, the relative suspension growth was 10% at the test substance concentration of 10 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 33μg/mL and above.
COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Remarks on result:

other: all strains/cell types tested

Further investigation showed that at concentrations 40 and 30 μg/mL in the first and second experiment, respectively the test substance already precipitated in the exposure medium. The test substance was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

First mutagenicity test

In the absence of S9-mix, the dose levels of 0.1 to 3 μg/mL showed no cytotoxicity. Therefore, the

dose levels of 0.1 and 0.3 μg/mL were not regarded relevant for mutation frequency measurement. The

dose levels of 9 to 13 μg/mL showed similar cytotoxicity. Therefore, the dose level of 13 μg/mL was not

regarded relevant for mutation frequency measurement.

In the presence of S9-mix, the dose levels of 0.1 to 30 μg/mL showed no cytotoxicity. Therefore, the

dose levels of 0.1 and 0.3 μg/mL were not regarded relevant for mutation frequency measurement. The

dose levels of 50 and 60 μg/mL showed similar cytotoxicity. Therefore, the dose level of 60 μg/mL was

not regarded relevant for mutation frequency measurement. The dose levels of 90 and 100 μg/mL were

not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were: Without S9-mix: 1,

3, 5, 7, 9, 11, 15 and 17 μg/mL exposure medium. With S9-mix: 1, 3, 10, 30, 40, 50, 70 and 80 μg/

mL exposure medium.

In the absence of S9-mix, the relative total growth of the highest test substance concentration of 17

μg/mL was reduced by 81% and of the dose level just below (15 μg/mL) by 90% compared to the total

growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the two highest test substance concentrations

were reduced by 67 and 66% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with

the test substance either in the absence or in the presence of S9-mix. The numbers of small and large

colonies in the test substance treated cultures were comparable to the numbers of small and large

colonies of the solvent controls.

Second mutagenicity test

To obtain more information about the possible mutagenicity of the test substance, a second mutation

experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence

of 8% (v/v) S9-mix with a 3 hour treatment period.

Evaluation of toxicity

In the absence of S9-mix, the dose levels of 12.5 to 20 μg/mL were not used for mutation frequency

measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 0.3 to 30 μg/mL showed no cytotoxicity. Therefore, the

dose levels of 0.3, 3 and 30 μg/mL were not regarded relevant for mutation frequency measurement.

The dose levels of 40 and 70 μg/mL showed similar cytotoxicity. Therefore, the dose level of 50 μg/mL

was not regarded relevant for mutation frequency measurement. The dose level of 85 μg/mL showed

an explainable high RSG and was not regarded relevant for mutation frequency measurement.

The dose levels selected to measure mutation frequencies at the TK-locus were: Without S9-mix: 0.03,

0.1, 0.3, 1, 3, 5, 8.5 and 10 μg/mL exposure medium. With S9-mix: 1, 10, 40, 60, 70, 75, 80 and 90

μg/mL exposure medium.

In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 86%

compared to the total growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was

reduced by 83% compared to the total growth of the solvent controls.

Evaluation of mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with

the test substance either in the absence or in the presence of S9-mix. The numbers of small and large

colonies in the test substance treated cultures were comparable to the numbers of small and large

colonies of the solvent controls.

Discussion

The spontaneous mutation frequencies in the solvent-treated control cultures were between the

minimum and maximum value of the historical control data range (See APPENDIX 3, Table 11).

The growth rate over the two-day expression period for cultures treated with ethanol was between 14

and 25 (3 hours treatment) and 77 and 95 (24 hours treatment) (See APPENDIX 2, Table 5, Table 6,

Table 8 and Table 9).

Mutation frequencies in cultures treated with positive control chemicals were increased by 9.2- and 9.5-

fold for MMS in the absence of S9-mix, and by 17- and 20-fold for CP in the presence of S9-mix, in

the first and second experiment respectively (See APPENDIX 1, Table 3 and Table 4). It was therefore

concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for

the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned

properly. In addition the observed mutation frequencies of the positive control substances were within

the acceptability criteria of this assay (See APPENDIX 3, Table 12).

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation

frequency in the first experiment. This result was confirmed in a repeat experiment with modifications

in the duration of treatment time.

In the presence of S9-mix, the test substance did not induce a significant increase in the mutation

frequency in the first experiment. This result was confirmed in an independent experiment with

modifications in the composition of the S9 concentration for metabolic activation.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test substance was not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Executive summary:

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation

test with L5178Y mouse lymphoma cells (with independent repeat) according to OECD Guideline No.

476 and EU Method B17. Therein, the effects of the test substance on the induction of forward mutations

at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells were observed. The test

was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix

induced by a combination of phenobarbital and B-naphthoflavone). The test substance was dissolved

in ethanol.

In the first experiment, the test substance was tested up to concentrations of 17 μg/mL and 80 μg/

mL in the absence and presence of 4% (v/v) S9-mix, respectively. The incubation time was 3 hours.

The test substance was tested up to cytotoxic levels of 90 and 67% in the absence and presence of

S9-mix, respectively. The test substance was tested beyond precipitating dose levels in the presence

of S9-mix. In the second experiment, the test substance was tested up to concentrations of 10μg/mL

and 90 μg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation times were 24 hours

for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. The

test substance was tested up to cytotoxic levels of 86 and 83% in the absence and presence of S9-

mix, respectively. The test substance was tested beyond precipitating dose levels in the presence of

S9-mix. The spontaneous mutation frequencies in the solvent-treated control cultures were between the

minimum and maximum value of the historical control data range and within the acceptability criteria

of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased

9.2- and 9.5-fold for MMS in the absence of S9-mix, and 17- and 20-fold for CP in the presence of S9-

mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix,

were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence

of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first

experiment. This result was confirmed in an independent repeat experiment with modifications in the

duration of treatment time. In the presence of S9-mix, the test substance did not induce a significant

increase in the mutation frequency in the first experiment. This result was confirmed in an independent

repeat experiment with modifications in the concentration of the S9 for metabolic activation. Hence, it

was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system

under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In the genotoxicity assays presented, the conclusions have been consistently negative (non-mutagenic). In vivo studies are considered to be not scientifically justified. From an assessment report (NICNAS) this consideration is supported.

Short description of key information:
This substance is non-genotoxic when tested in vitro.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

This substance is non-genotoxic when tested in vitro and in vivo, and so is not classified as a mutagen.