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Genetic toxicity in vitro

Description of key information

A battery of in vitro tests was performed with THEIC to determine its genotoxic potential (Ames test, chromosome aberration test, sister chromatid exchange, mouse lymphoma tests, each with and without metabolic activation). In each of these studies no genotoxic potential was detected and no cytotoxicity was observed.

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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment, although not GLP compliant. The study was conducted according to an internationally accepted technical guideline in a recognized contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1983
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male Sprague Dawley rats treated by single intraperitoneal injection with Aroclor 1254 (500 mg/kg bw) for enzyme induction. Aroclor 1254 was administered 5 days prior to sacrifice.
Test concentrations with justification for top dose:
Experiment 1 (Standard Plate Test, without and with metabolic activation (S9 mix), Doses: 0; 20; 100; 500; 2500 and 5000 μg/plate
Experiment 2 (Pre-incubation Test, without and with metabolic activation (S9 mix), Doses: 0; 20; 100; 500; 2500 and 5000 μg/plate
Vehicle / solvent:
aqua dest.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (disolved in DMSO)
Remarks:
Positive control substance for all tests with metabolic activation (S9 mix)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine for TA 1535 & TA 100; 4-nitro-o-phenylendiamine for TA 98; 9-aminoacridine chloride monohydrate for TA 1537 (each positive control substance disolved in DMSO)
Remarks:
Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.
Details on test system and experimental conditions:
Standard Plate Tests were performed in Experiment 1, Pre-incubation Tests in Experiment 2.
Both experiments were conducted without and with metabolic activation (S9 mix)

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

With metabolic activation (S9 mix):

2-aminoanthracene:
- 10 μg/plate, dissolved in DMSO: for the strains: TA 1535, TA 100, TA 1537, TA 98

Without metabolic activation (S9 mix):

N-methyl-N'-nitro-N-nitrosoguanidine:
- 5 μg/plate, dissolved in DMSO: for the strains: TA 1535, TA 100

4-nitro-o-phenylendiamine:
- 10 μg/plate, dissolved in DMSO: for the strain: TA 98

9-aminoacridine chloride monohydrate:
- 100 μg/plate, dissolved in DMSO: for the strain: TA 1537


Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control),
- dose-response relationship,
- reproducibility of the results.
Statistics:
Statistical analysis of the results was not reported. Obviously it was not considered to be necessary.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was completely soluble in the vehicle, aqua dest.

Contamination was noted only in one plate which was one of the TA 100 without metabolic activation at 20 µg/plate. In all other solvent control, test substance treated or positive control plates contamination was not evident.
Conclusions:
Interpretation of results :
negative without and with metabolic activation (S9 mix)
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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to information in OECD SIDS (2002)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: essential amino acid requiring strain
Metabolic activation:
with and without
Metabolic activation system:
S9mix from Phenobarbital and 5,6-benzoflavone induced rat liver microsomes
Test concentrations with justification for top dose:
0; 156; 313; 625; 1250; 2500; 5000 µg/plate, each concentration with and without metabolic activation (S9 mix).
Vehicle / solvent:
Water for injection
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
OECD SIDS confirms "negative control groups" and gives a vehicle, but it does not distinguish between "negative controls", "solvent/vehicle controls" and "true negative controls"
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control substance for all strains in all tests with metabolic activation (S9 mix).
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
OECD SIDS confirms "negative control groups" and gives a vehicle, but it does not distinguish between "negative controls", "solvent/vehicle controls" and "true negative controls"
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide for TA100, TA98 & WP2 uvrA; Sodium azide for TA1535; 9-Aminoacridine hydrochloride for TA1537
Remarks:
Positive control substances for tests without metabolic activation (S9 mix)
Details on test system and experimental conditions:
Number of replicates: 2
Plates per test: 3
Procedure: Pre-incubation

Evaluation criteria:
The result was designated "mutagenic"  when at least a two-fold increase over the control, and a dose response-trend  or reproducibility were observed.
Statistics:
No statistic analysis.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Interpretation of results:
negative without and with metabolic activation (S9 mix)
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
of 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Types and identity of media:
Reduction of spontaneous mutants by growth for 1 day in RPMI 1640-HAT medium supplemented with hypoxanthine, aminopterin and thymidine.
Then recovery period of 2 days in RPMI 1640 medium supplemented with hypoxanthine and thymidine.
Thereafter return to normal complete culture medium, i.e. RPMI 1640 medium supplemented with:
penicilin, streptomycin, sodium pyruvate, amphotericin and with 3% horse serum during 4 h treatment and 15% horse serum during 24 h treatment
Routine cell culturing at 37±1.5°C with 4.5% CO2 in air.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from male Wistar rats treated on 3 consecutive days by i.p. injection with phenobarbital and by oral gavage with β-naphthoflavone (80 mg/kg/day, each) for enzyme induction. The concentration of S9 mix in final test medium was 5% (v/v).
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TESTING (suspension growth relative to that of vehicle controls)
Test concentrations at 4 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9):
0, 20.5, 40.9, 81.9, 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL

MUTATION TESTS
Experiment 1, 4 h exposure (–/+S9):
Exposure concentrations: 81.9, 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL
Mutant phenotype determination at: 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 81.9, 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL
Mutant phenotype determination at: 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL

Experiment 2, 4 h exposure (+S9):
Exposure concentrations: 81.9, 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL
Mutant phenotype determination at: 163.8, 327.5, 655.0, 1310.0 and 2620.0 µg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
In the absence of confounding factors, four analysable concentrations up to 5000 µg/mL or 10 mM for freely soluble test substances are the guideline requirement for metaphase analysis. In the present study, relevant cytotoxicity was not evident, THEIC has been well soluble in the vehicle, de-ionised water, and variations in osmolality and pH between vehicle control and high dose culture media were within acceptable limits. Hence, there were no confounding factors limiting the choice of test concentrations for metaphase analysis.
Vehicle / solvent:
deionised tap water.

Justification for choice of solvent/vehicle:
The test substance has been well soluble in the chosen vehicle thus facilitating maximum exposure to the test substance.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised tap water (10% final concentration in the medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control substance for tests without metabolic activation (-S9) in Experiments 1 and 2 Migrated to IUCLID6: 4h exposure: 19.5 µg/mL; 24 h exposure: 13.0 µg/mL, dissolved in nutrient medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised tap water (10% final concentration in the medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive control substance for tests with metabolic activation (+S9) in Experiments 1 and 2 Migrated to IUCLID6: 3.0 µg/mL and 4.5 µg/mL, dissolved in 0.9% saline
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1: 4 h exposure with (+S9) and without (–S9) metabolic activation
Experiment 2: 4 h exposure with (+S9) and 24 h exposure without metabolic activation (–S9)

- Selection time: At 48 h after the end of exposure exposure to the selection agent trifluorothymidine (TFT)
then allowing 11-12 days for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 cultures at each concentration,
[from each culture one vial for assessment of growth in suspension, two 96-well plates for assessment of cloning efficiency
and two 96-well plates for assessment of mutant potential].

NUMBER OF CELLS EVALUATED: ca. 4000 cells/well x 192 wells = ca. 800000 cells per experimental group and culture

DETERMINATION OF CYTOTOXICITY: Relative total growth and Relative suspension growth; (in preliminary toxicity test Relative suspension growth)
Evaluation criteria:
Mutagenic response of test material was considered to be evident if the mutant frequency of any test concentration reproducibly was higher than the sum of the concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF = 126 x 10^–6, Moore et al. 2006, detailed reference see below). A mutagenic response was considered to be reproducible if it occurred in both concomitant cultures. Relevant increases in mutation frequency should be dose-dependent. However, historical variability of mutation frequency in solvent controls was duely accounted for.

A final decision on mutagenicity was taken by the study director case by case accounting also for reproducibility, dose-relationship and the degree of mutant frequency increase.

In case of evident mutagenicity, the ratio of small versus large colonies is used to distinguish point mutations from clastogenic effects. If the increase in mutation frequency is accompanied by a reproducible and dose dependent increase in the ratio of small to large colonies clastogenic effects are indicated.

Reference for GEF:
Moore MM, Honma M, Clements J, Bolcsfoldi G, Burlinson B, Cifone M, Clarke J, Delongchamp R, Durward R, Fellows M, Gollapudi B, Hou S, Jenkinson P, Lloyd M, Majeska J, Myhr B, O’Donovan M, Omori T, Riach C, San R, Stankowski Jr. LF, Thakur AK, Van Goethem F, Wakuri S and Yoshimura I (2006). Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis 47, 1-5.
Statistics:
The data were analysed by linear regression (least squares) to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 (SYSTAT Software, Inc., Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for test substance treated groups was compared with the solvent control groups. p-values < 0.05 were considered to indicate a statistically significant trend. However, both, biological relevance and statistical significance were given due consideration.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Experiment 1, 4 h exposure
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Experiment 2, 4 h exposure
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Experiment 2, 24 h exposure. One toxicologically irrelevant outlier showing considerable increase in mutation frequency. Considered to be incidental, as this is confined to one low test concentration (163.8 µg/mL) in Culture 1 of the 2 replicate cultures.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
One irrelevant outlier showing 37.7% relative total growth. Considered to be incidental, as this is confined to one low test concentration (163.8 µg/mL) in Culture 1 of the 2 replicate cultures.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In Culture 1 of the two cell cultures used in the present study, some of the acceptance criteria of the International Workshop on Genotoxicity Test Procedures (IWGT) in general were not met in the positive controls, although these did induce distinct increases in mutation frequency exceeding the thresholds for an effect and meeting the historical reference range for positive controls. This was related to the fact that most of the induced colonies were large ones although the positive controls are known to induce mainly clastogenic effects. Hence culture 1 could not distinguish point mutations from clastogenic effects. This did not compromise the results or validity of the study, because mutagenicity of THEIC was not evident, positive control data of Culture 2 in general met the IWGT recommendations and the positive control data of both cultures met the requirements of OECD 476.
Conclusions:
Interpretation of results :
negative without and with metabolic activation (-/+S9)
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to information in OECD SIDS (2002)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster Lung cells (CHL/IU)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver, treated with Phenobarbital and 5,6-benzoflavone for enzyme induction
Test concentrations with justification for top dose:
0, 653, 1306, 2612 µg/mL for short-term treatment (6 h), without and with metabolic activation (S9).
0, 653, 1306, 2612 µg/mL for continuous treatment (24 h), without metabolic activation (S9).
Vehicle / solvent:
physiol. saline
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
OECD SIDS confirms "negative control groups" and gives a vehicle, but it does not distinguish between "negative controls", "solvent/vehicle controls" and "true negative controls" Migrated to IUCLID6: without metabolic activation
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
OECD SIDS confirms "negative control groups" and gives a vehicle, but it does not distinguish between "negative controls", "solvent/vehicle controls" and "true negative controls" Migrated to IUCLID6: with metabolic activation
Details on test system and experimental conditions:
For continuous treatment, cells were treated for 24 h without S9.
For short-term treatment, cells were treated for 6 h with and without S9 and cultivated with fresh media for 18 h.

Plates per test: 2
Evaluation criteria:
The results were considered to be:
- negative, if the incidence was less than  4.9%,
- equivocal, if it was between 5.0 and 9.9%, and
- positive, if it was more than 10.0%.
Statistics:
No statistic analysis. 
Key result
Species / strain:
other: Chinese Hamster Lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
According to the OECD SIDS Initial Assessment Profile, the maximum concentration of 2612 µg/mL tested in the present study represented the maximum concentration with no apparent cytotoxic effect in short-term (6 hours) and continuous treatments (24 hours).

Structural chromosomal aberrations and polyploidy were not induced up to the maximum concentration tested under conditions of short-term treatment(6h)with and without an exogenous metabolic activation system and under conditions of continuous treatment (24h) without metabolic activation. 

.

Conclusions:
Interpretation of results :
negative Without and with metabolic activation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the negative results attained in all in vitro genotoxicity studies, THEIC is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].