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Toxicological information

Genetic toxicity: in vitro

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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:
13/01/2012-24/05/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP comparable to OECD guideline
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Principles of method if other than guideline:
/
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Brominated epoxy having epoxy equivalent of 400gr/eq
- Substance type: mixture of oligomers
- Physical state:pale yellow solid
- Analytical purity: >99%
- Lot/batch No.: B21E-86
- Expiration date of the lot/batch:30/05/2015
- Storage condition of test material:room temperature in the dark

Method

Target gene:
/
Species / strain
Species / strain / cell type:
lymphocytes: human
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 treatment
Test concentrations with justification for top dose:
Experiment 1 (4 hour exposure without S9-mix): 80, 160, 320, 640, 1280 and 2560 μg/ml
Experiment 1 (4 hour exposure with S9-mix): 10, 20, 40, 80, 120 and 160 μg/ml
Experiment 2 (24-hour exposure without S9-mix): 40, 80, 120, 160, 320 and 640 μg/ml
Experiment 2 (24-hour exposure with S9-mix): 10, 20, 40, 80, 120 and 160 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (DMSO was selected as the solvent because the test item was soluble at 50mg/ml
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: in medium

DURATION
-preincubation: 48 hours
- Exposure duration:experiment 1: 4 hours, experiment 2: 24hours
- 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):26 hours

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):when the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS:1 (experiment 1), 2 (experiment 2).

NUMBER OF CELLS EVALUATED:2000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
MITOTIC INDEX: A total of 200 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
SCORING OF CHROMOSOME DAMAGE: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. It the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome abberations were reviewed as necessary by a senior cytogenticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical test may be applied in order to required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:All of the vehicle control cultures had frequencies of cells with chromosome abberations within the expected range.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1. PRELIMINARY TOXICITY TEST:Dose selection for experiments 1 and 2 was based on toxicity, in relation to the precipitate observed, in all of the exposure groups.

2. EXPERIMENT 1: The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

3. EXPERIMENT 2:

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

INTRODUCTION

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method was designed to be compatible with the OECD guidelines for testing of chemicals (1997) No 473 and Method B10 (EC) and US EPA OPPTS 870.5375 and is acceptable to the Japanese New Chemical Substance Law (METI)

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels (selected from the dose levels tested below), together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 -hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20 -hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20 -hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in Experiments 1 and 2 were selected using data from the preliminary toxicity test and were as follows:

 Experiment  Group  Final concentration of test item (µg/ml)    
 1  4(20)-hour without S9  80, 160, 320, 640, 1280, 2560    
 1  4(20)-hour with S9 (2%)  10, 20, 40, 80, 120, 160    
 2  24 -hour without S9  40, 80, 120, 160, 320, 640    
 2 4(20)-hour with S9 (1%)   10, 20, 40, 80, 120, 160