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Genetic toxicity in vitro

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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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Target gene:
/
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
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.
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'.

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.

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    
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:
13/01/2012-13/02/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP comparable to OECD guideline
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
/
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
hisD, hisG, hisC, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate
Experiment 1: 50, 150, 500, 1500, 5000μg/plate
Experiment 2: 15, 50, 150, 500, 1500, 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: fully soluble in DMSO, 100mg/ml in aceton
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation

Migrated to IUCLID6: strain WP2uvrA, TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation

Migrated to IUCLID6: strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation

Migrated to IUCLID6: strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for strain TA100, TA1535, Ta1537, WP2uvrA
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: strain TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:/
- Exposure duration:48h
- Expression time (cells in growth medium):48h




NUMBER OF REPLICATIONS:3

Evaluation criteria:
1. A dose related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accomponied by an out-of-historical range response).
Statistics:
/
Species / strain:
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item, Brominated Epoxy having epoxy equivalent of 400gr/eq, was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities, OECD 471, B13/B14 (EC), OPPTS guidelines (EPA).

Methods Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Brominated epoxy having epoxy equivalent of 400gr/eq, using both the Ames plate incorporation and pre-incubation methods at up to six dose elvels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was50 to 5000µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended (15 to 5000µg/plate) to allow for the change in test methodology in the second experiment from plate incorporation to pre-incubation.

Results. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. A test item precipitate (white and particulate in appearance) was noted at and above 1500µg/plate, this observation did not prevent the scoring of revertant colonies.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2013- March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and appropriate testing guidelines
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:
Exposure Group Final concentration of test item (µg/mL)
4-hour without S9 5, 10, 20, 40, 60, 80
4-hour with S9 (2%) 5, 10, 20, 40, 60, 80
4-hour without S9 10, 20, 40, 60, 70, 80
4-hour with S9 (2%) 10, 20, 40, 60, 70, 80

Vehicle / solvent:
Dimethyl sulphoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methane sulphonate (EMS) was used at 500 µg/mL and 750 µg/mL as the positive control in the 4-hour cultures without S9. Dimethyl benzanthracene (DMBA) at 1.0 and 2.0 µg/mL was used as the positive control in cultures with S9.
Details on test system and experimental conditions:
Chinese hamster (V79) cells were treated with the test item at six dose levels, in duplicate, together with the vehicle (dimethyl sulphoxide) and the positive controls (EMS and DMBA) in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test, i.e. In in Experiment 1, a 4 hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9).
In Experiment 2, the exposure conditions were repeated to confirm the result of the first experiment using revised dose ranges. The dose range of the test item was selected based on the results of a preliminary cytotoxicity test.


Evaluation criteria:
The test item is considered to be mutagenic if there is a reproducible dose-related increase in the mutation frequency where at least a threefold increase in the mutant frequency over the vehicle control value is observed. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
A test item producing neither a dose-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations is not sufficient to evaluate the test item as a mutagen. See also Attachment no. 1 below.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The vehicle (dimethyl sulphoxide) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.
The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment

The test item was shown to be markedly toxic in the presence of S9 but non-toxic in the absence of S9 in the Preliminary Cytotoxicity Test. The dose range for the main experiments were therefore influenced by the presence of precipitate and the toxicity seen in the presence of S9. The maximum dose selected for both exposure groups of Experiment 1 was limited to 80 µg/mL where optimum exposure to the test item was considered to occur and it was the lowest precipitating dose level with true precipitate for the exposure group in the absence of S9. The exposure conditions of Experiment 1 were repeated for Experiment 2 with a revised dose range and the marginal increases in mutant frequency seen in Experiment 1 were not repeated confirming the absence of any response.
 
Precipitate observations did vary slightly between the different experiments and this is considered to be due to the highly subjective nature of making these observations. However, both precipitating and non-precipitating dose levels have been tested in the absence of S9 and in the presence of S9 optimum toxicity has been achieved meaning that the test item has been adequately tested and the requirements of the test guideline have been met.
 
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. Brominated Epoxy was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

The purpose of this study was to assess the potential mutagenicity of Brominated Epoxy, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

  Methods: Chinese hamster (V79) cells were treated with the test item at six dose levels, in duplicate, together with the vehicle (dimethyl sulphoxide) and positive controls in the presence and absence of an S9 metabolic activation system. 

Four treatment conditions were used for the test: in Experiment 1, a 4‑hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the exposure conditions were repeated to confirm the result of the first experiment using revised dose ranges.

 The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows: 

Exposure Group

Final concentration oftest item(µg/mL)

4-hour without S9

5, 10, 20, 40, 60, 80

4-hour with S9 (2%)

5, 10, 20, 40, 60, 80

4-hour without S9

10, 20, 40, 60, 70, 80

4-hour with S9 (2%)

10, 20, 40, 60, 70, 80

 

 Results: The vehicle (dimethyl sulphoxide) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

 The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment

 

Conclusion

Bominated Epoxy was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Brominated Epoxy does not show any genetic toxicity proporties in all studies performed.


Justification for selection of genetic toxicity endpoint
Genetic toxicity was not observed in Brominated epoxy.

Short description of key information:
Brominated Epoxy did not show any potential for being a genetic toxicant in any of the 3 in vitro stuides conducted:

1. AMES (Thompson 2012)- Brominated Epoxy having epoxy equivalent of 400gr/eq, was considered to be non-mutagenic under the conditions of this test.

2. In vitro Chromosome aberration test (Bowles 2012) - Brominated epoxydid 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.

3. In vitro V79 HPRT Gene Mutation Assay- Bominated Epoxy was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test item did not meet the criteria for classification as a mutagen according to EU labelling regulations Commision Directive 2001/59/EC or the globally harmonised system of classification and labelling of chemicals.