Hexanoic acid, 3,5,5-trimethyl-, 2,2,6,6-tetramethyl-1-[2-[(3,5,5-trimethyl-1-oxohexyl)oxy]ethyl]-4-piperidinyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames tests, an HPRT assay and a chromosome aberration test in CHO cells and V79 cells, respectively, was performed according GLP and OECD guideline 471, 473 and 476to evaluate the mutagenic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. Therefore, the substance is not considered to be mutagenic under the conditions of these tests.

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

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented, according to OECD guidelines and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital i.p. and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

33 μg - 5 200 μg/plate (SPT)
33 μg - 5 200 μg/plate (PIT)

Vehicle / solvent:

Due to the limited solubility of the test substance in ultrapure water, acetone was used as
vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and
for which historical control data are available

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Remarks:

Without S9 mix

Details on test system and experimental conditions:

DETAILS ON TEST SYSTEM
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours
NUMBER OF REPLICATIONS: in triplicate
NUMBER OF CELLS EVALUATED: all revertants / colonies counted
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and
E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA
1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either
without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control
data range under all experimental conditions in at least two experiments carried out
independently of each other.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY: Precipitation of the test substance was found depending on the test conditions from about 1 000 μg/plate onward.
TOXICITY: No bacteriotoxic effect was observed under all test conditions

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of 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

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented, according to OECD guideline and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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 with Earle's salts) containing a L-glutamine source supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericin B (250 μg/mL)
During exposure to the test substance in the presence of S9 mix MEM medium was used without FCS supplementation.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
4-hour exposure, 18-hour sampling time, without S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50; 100 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50; 100 μg/mL

2nd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50; 100 μg/mL
18-hour exposure, 28-hour sampling time, without S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50; 100 μg/mL
4-hour exposure, 28-hour sampling time, with S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50; 100 μg/mL

Vehicle / solvent:

Acetone 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4h +/- S9 mix experiment I, 18h and 28h without S9 mix and 4h with S9 mix experiment II
- Expression time (cells in growth medium): after 4h exposure 14-24h, after 18h or 28h exposure 0h
- Fixation time (start of exposure up to fixation or harvest of cells): after 4h exposure 18-28h, 18h and 28h exposure and fixation direct thereafter

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments, samples / cultures in triplicate

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases will be scored per culture

DETERMINATION OF CYTOTOXICITY
- Method: slides will be evaluated for mitotic index and cell numbers, 1000 cells per culture will be scored and values will be expressed as a percentage of the solvent controls

Evaluation criteria:

The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells
with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle
control value and the historical negative control data range (see Appendix 5).
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not
statistically significant increased above the concurrent negative/vehicle control value and
is within the historical negative control data range

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system
(BASF SE). The proportion of metaphases with structural aberrations was calculated for each
group. A comparison of each dose group with the negative control group was carried out
using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-
Holm corrected versus the dose groups separately for each time and was performed
one-sided. If the results of this test are statistically significant compared with the respective
vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.

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:

valid

Positive controls validity:

valid

Additional information on results:

Osmolarity and pH values were not influenced by test substance treatment.
The test substance was poorly soluble in culture medium. Test substance precipitation in
culture medium at the end of exposure period was observed at 100 μg/mL in all experimental
parts in the absence of S9 mix and from 50 μg/mL onward in all experimental parts in the
presence of S9 mix, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

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:

2014/2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented, according to OECD guideline and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hpgrt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams F12
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL
with S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

2nd Experiment
without S9 mix (4-hour exposure period)
0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL
with S9 mix (4-hour exposure period)
0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 3 days (4 hour treatment) or 2 days (24 hour treatment) at 37°C
- Selection time (if incubation with a selection agent): 6-7 days in TG-medium
- Fixation: At the end of the selection period, the medium will be removed and the remaining colanies will be fixed with methanol, stained with Giemsa and counted

SELECTION AGENT (mutation assays): TG-medium

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
 Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent
negative control values and our historical negative control data range (see Appendix 6).
 Evidence of the reproducibility of any increase in mutant frequencies.
 A statistically significant increase in mutant frequencies and the evidence of a doseresponse
relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e.
15 mutants per 106 clonable cells) or isolated statistically significant increases without a
dose-response relationship may indicate a biological effect but are not regarded as sufficient
evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
 The corrected mutation frequency (MFcorr.) in the dose groups is not statistically
significantly increased above the concurrent negative control and is within our historical
negative control data range.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a
dose-related increase of mutant frequencies. The number of mutant colonies obtained for the
test substance treated groups was compared with that of the respective vehicle control
groups. A trend is judged as statistically significant whenever the one-sided p-value
(probability value) is below 0.05 and the slope is greater than 0. However, both, biological
and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

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:

valid

Positive controls validity:

valid

Additional information on results:

The test substance was poorly soluble in culture medium. In this study, in the absence and
the presence of metabolic activation no cytotoxicity was observed up to the highest required
concentration. Thus, concentrations at the border of test substance solubility in culture
medium were investigated for the induction of gene mutations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro assays were performed to evaluate the genotoxicity of the test substance. At first, the material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test item was applied at concentrations of 33 μg - 5000 μg/plate (standard plate test) and 33 μg - 5000 μg/plate (pre-incubation test) in presence and absence of a metabolic activation system (S9 mix). Precipitation of the test substance was found depending on the test conditions from about 1 000 μg/plate onward. No bacteriotoxic effect was observed under all test conditions.

The substance was assessed for its potential to induce gene mutations at thehypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and beta-naphthoflavone induced rats (exogenous metabolic activation) at concentrations up to 50 ug/ml. The test substance was poorly soluble in culture medium. In this study, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration. Thus, concentrations at the border of test substance solubility in culture medium were investigated for the induction of gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

The substance was assessed for its potential to induce structural chromosome aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation) at concentrations up to 100 ug/ml. A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures where only 50 metaphases were scored due to clearly increased aberration rates. The test substance was poorly soluble in culture medium. In this study, no cytotoxicity indicated by distinctly reduced cell numbers or mitotic rates was observed up to the highest applied test substance concentrations which were at the border of test substance solubility in culture medium. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Short description of key information:
An Ames tests, an HPRT assay and a chromosome aberration test in CHO cells and V79 cells, respectively, was performed according GLP and OECD guideline 471, 473 and 476to evaluate the mutagenic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. Therefore, the substance is not considered to be mutagenic under the conditions of these tests.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.