Tris(2-hydroxyethyl)-1,3,5-triazinetrione

Endpoint summary

Key value for chemical safety assessment

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.

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

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

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)

Reference 2

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

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)

Reference 3

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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

of 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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)

Reference 4

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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

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:

mammalian cell line, 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

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].