Reaction mass of 1,2,2,6,6-pentamethylpiperidin-4-yl hexadecanoate and 1,2,2,6,6-pentamethylpiperidin-4-yl octadecanoate

Genetic toxicity: in vitro

Administrative data

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:

08 May 2012 - 13 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to internationally accepted guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Official notice of MHLW, METI and MOE (31 March 2011), YAKUSHOKUHATSU 0331 No 7, SEIKYOKU No 5, KANPOKIHATSU No 110331009.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,2,2,6,6-pentamethylpiperidin-4-yl hexadecanoate and 1,2,2,6,6-pentamethylpiperidin-4-yl octadecanoate

EC Number:

700-792-4

Molecular formula:

C26H51NO2, C28H55NO2

IUPAC Name:

Reaction mass of 1,2,2,6,6-pentamethylpiperidin-4-yl hexadecanoate and 1,2,2,6,6-pentamethylpiperidin-4-yl octadecanoate

Details on test material:

- Name of test material (as cited in study report): T-1640L
- Physical state: white - pale yellow solid
- Analytical purity: 98.7%
- Purity test date: 20 May 2011
- Lot/batch No.: 10121
- Expiration date of the lot/batch: 12 April 2013
- Stability under conditions of the test: stable
- Storage condition of test material: ca. 4°C, in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix derived from rat livers

Test concentrations with justification for top dose:

First test:
46.9, 78.2, 130.4, 217.3, 362.2, 603.7, 1006.1, 1676.8, 2794.7 and4657.9 μg/mL in absence of S9 mix & in presence of S9 mix. Appropriate toxicity profile was not achieved, addtional test conducted at concentrations as below:
In absence of S9 mix: 4, 16.9, 28.2, 46.9, 78.2, 130.4, 217.3, 362.2, 603.7, 1006.1, 1676.8, 2794.7 & 4657.9 μg/mL
In presence of S9 mix: 125, 250, 500, 750, 1000, 2000, 2750, 3000, 3500, 4000 and 4657.9 μg/mL


Second test:
30, 50 and 60 μg/mL in absence of S9 mix
250, 500 and 1000 μg/mL in presence of S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dehydrated acetone
- Justification for choice of solvent/vehicle: T1640L soluble at 465.79 mg/mL (1 M) in the vehicle, dilution to 279.47 mg/mL no precipitate observed

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation;

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hr (presence of S9 mix), 21 hr (absence of S9 mix)
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: statistical evaluation of aberrant metaphase cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other:

OTHER:

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range. The test substance is considered to cause a positive response if the following conditions are
met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentrations.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a
log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002). The following
model was used:
p = C + ((1 – C)/(1 + exp{– intercept – slope ln(conc)})

p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations. The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.0) which was developed in SAS (SAS INSTITUTE 2002).

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that T-1640L has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described. The test substance T-1640L has, however, shown a statistically significant
increase in numerical aberrations in the form of polyploidy, in the absence of S9 mix only, in this in vitro cytogenetic test system, under the conditions described.