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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

TEA did not cause gene mutations in Salmonella typhimurium and Escherichia coli (Ames test), nor were chromosomal aberrations or sister chromatid exchanges induced in Chinese hamster ovary cells observed. An in vitro gene mutation assay (mouse lymphoma (L5178Y TK+/-) forward gene mutation assay) was also negative. All tests were performed in the absence and presence of metabolic activation.

Link to relevant study records

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Method: other: after Galloway, S.M. et al.: Environ. Mutagen. 7, 1-51
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
1510 - 4030 µg/mL (without S9)
6040 - 10100 µg/mL (with S9)
Doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment.
Vehicle / solvent:
Water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: triethylenemielamine, mitomycin C. or cyclophosphamide
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537 or TA97, TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat and hamster metabolic activation
Test concentrations with justification for top dose:
up to 10 mg/plate, no further data available
Vehicle / solvent:
Distilled water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
TA98
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA97 and TA1537
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains
Details on test system and experimental conditions:
Type: Ames test
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
but testes up to limit concentrations
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:
not specified
Remarks:
but testes up to limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
but testes up to limit concentrations
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:
not specified
Remarks:
but testes up to limit concentrations
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:
not specified
Remarks:
but testes up to limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot # 000STD77L0



Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 50, 100, 250, 500, 1000 and 1500 µg/mL
Vehicle / solvent:
distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl methanesulfonate (MMS) was used at concentrations of 10 and 15 µg/ml of culture medium in the nonactivation assay while 20-methylcholanthrene (20-MCA) was used at concentrations ranging from 1.0-5.0 µg/ml.
Details on test system and experimental conditions:
Two independent mutation assays were conducted to assess the mutagenic potential of the test material.

Treatment Procedure
Cells from logarithmically growing stock cultures (maintained in an orbital shaker at 37C) were treated in individual 50 ml culture tubes. To these cells, 4 ml of either S9 mix or medium was added along with the test material. Following the addition of the test compounds, the tubes were incubated for approximately 4 hours at 37°C in a roller drum (approximately 25-35 orbits/minute). At the end of the incubation period, the cells were pelleted, rinsed with medium and resuspended. The tubes were returned to the roller drum and maintained at 37°C during a standard expression period of two days.

Toxicity Assay
This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. The cells were treated with various concentrations of the test material (one replicate per dose) in the absence and presence of S9. At approximately 24 hours after treatment (day 1), the test cultures were counted. If the treated cells failed to multiply to a density of 4 x 10E05 on the first day following treatment, the culture was returned to the incubator without any dilution. On day 2, cultures were again counted for cell density. From these cell counts, the following indices were calculated:
Day 1 SG = Suspension Growth over first day
Day 2 SG = Suspension Growth during second day
CSG = Cumulative Suspension Growth during first 2 days
Day 2 RSG (%) = Cumulative relative suspension growth over first two days

Gene Mutation Assay
Each dose level was set up in duplicate from the time of treatment until the completion of the assay with the exception of positive controls where only one replicate was used. At the end of treatment, cells were returned to the incubator for phenotypic expression. At 24 hours following treatment (day 1), the test cultures were counted and diluted to a concentration of approximately 3 x 10E05 cells/ml. If the treated cells failed to multiply to a density of 4 x 10E05 cells/ml on the first day following treatment, the culture was returned to the incubator without any dilution. On day 2 (48 hours following treatment) cultures were again counted and treatment levels with desired levels of toxicity were selected for cloning. Cultures with < 10% day 2 RSG were not cloned. A total sample size of 3 x 10E06 cells from each culture was suspended in cloning medium with trifluorothymidine (TFT) and plated into three petri dishes (100 mm), allowed to gel for approximately 15 minutes at 0-6°C, and returned to the incubator for approximately 13 days to allow for mutant colony formation. The cloning efficiency was determined by serially diluting the sample in cloning medium without 1 µg/ml TFT and then plating the cells into three petri dishes (100 mm) at a concentration of approximately 200 cells per dish. The dishes were returned to the incubator for approximately 13 days before counting the number of colonies per dish.
An image analyzer (LAI High-Resolution Colony Counting System, Loats Associates, Inc., Westminster, Maryland) was used to count and size colonies. The separation of small and large colonies was determined by inspection of colony sizing histograms of each culture. Mutant colonies form a bimodal distribution and the cutoff between the two distributions was set manually.
The parameter relative total growth (RTG) was used to determine the cytotoxicity of various treatments.
Initially, plates for cloning efficiency were counted and the RTG values calculated. Only those cultures with RTG values ≥ 10% were counted for mutants. However, one exception occurred in the positive control cultures in the presence of S9 in the initial gene mutation assay where the RTG values were less than 10% and the mutant plates were evaluated. These plates were deemed suitable for counting in the initial assay and the data was confirmed in the repeat assay using a lower concentration of the positive control.
Evaluation criteria:
Mutant frequencies were evaluated based upon biological significance criteria. The test chemical was considered positive when the conditions listed below were met:
a) the average mutant frequency in at least one dose level of the treated cultures (resulting in ≥ 10% relative total growth) was 90 x 10-6 above the average of the concurrent solvent controls (assuming these to be in the range of 35-140 x 10-6).
b) there was a positive dose related linear trend. This was tested using a linear trend test at alpha = 0.05, provided the above criteria was met.

The test material was considered negative in this assay if the following condition was met:
a) there was no evidence of increase in mutant frequency at RTG values ≥ 10%.

The test material was considered equivocal in this assay if the following conditions were met:
a) there was a significant increase in mutant frequency only at RTG values > 10% and < 20%.
b) there was no evidence of increase in mutant frequency at RTG values ≥ 20%.
Statistics:
The activation and non-activation assays were considered independent assays with their own solvent and positive controls. For an assay to be considered acceptable, (1) the mutation frequency of positive controls should have been significantly higher than the solvent controls and (2) the mutant frequency of the solvent controls should have been within 35-140 x 10-6. The solvent controls must have had an average absolute cloning efficiency between 65-120% and a cumulative suspension growth greater than eight.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In a preliminary toxicity assay, the test material was evaluated at concentrations ranging from 5.9 to 1500 µg/mL. In the absence of S9, little to no toxicity was observed with day 2 relative suspension growth (RSG) values ranging from 81.2 to 124.2%. In the presence of S9, the corresponding day 2 values ranged from 80.7 to 101.5%.

There were no biologically significant increases in the mutant frequencies in the test material-treated cultures compared to the solvent control cultures in either absence or presence of S9. The results of this in vitro mouse lymphoma (L5178Y TK+/-) forward gene mutation assay with triethanolamine indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence and presence of an externally supplied metabolic activation (S9) system.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method: other: after Galloway, S.M. et al.: Environ. Mutagen. 7, 1-51
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
100 - 2520 µg/mL (without S9)
330 - 10100 µg/mL (with S9)
In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/mL or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.
Vehicle / solvent:
Water, dimethyl suifoxide (DMSO), ethanol, or acetone, in that order of preference.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

TEA was tested in the Ames reverse mutation assay using S. typhimurium strains TA 1535, TA 1537, TA 97, TA 98 and TA 100 at a concentration up to 10000 µg/plate with and without metabolic activation. Treatment with TEA was not associated with reverse mutations in any of the strains tested (Mortelmans, 1986). In another bacterial mutation assay using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli strains WP2 and WP2 uvrA, TEA was tested at concentrations up to 4000 µg/plate with and without metabolic activation. In this assay, TEA was not genotoxic in all the strains tested (Dean, 1985). TEA was also tested negative in a bacterial mutation study using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli strains WP2 and WP2 uvrA with and without metabolic activation at concentrations up to 2000 µg/plate (TSCATS, 1989). In a fourth mutation assay, using S. typhimurium strains TA 98 and TA 100 and E. coli strain WP2, TEA was tested at concentrations up to 20000 µg/plate with and without metabolic activation. In this assay, TEA was also tested negative (Innoue, 1982). Induction of chromosomal aberrations and sister chromatid exchanges was investigated in Chinese hamster ovary cells, exposed to concentrations up to 10100 µg/mL (which induced cytotoxicity). All tests were negative in the absence as well as the presence of metabolic activation (Galloway, 1987). TEA was also negative in the in vitro mouse lymphoma (L5178Y TK+/-) forward gene mutation assay (The Dow Chemical Company, 2010). Two independent assays at concentrations ranging from 50 to 1500 mg/mL in the absence and presence of an externally supplied metabolic activation (S9) system were performed. The highest concentration tested was the limit dose of 10 mM.

Based on the available information, IARC (2000) concluded that TEA was not mutagenic to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA 1538 in the presence or absence of exogenous metabolic activation in a number of studies. TEA did not induce mutations in Escherichia coli WP2 uvrA and WP2 try- in the presence or absence of exogenous metabolic activation in two studies. In a single study, TEA was not mutagenic to Bacillus subtilis strains carrying uvrA or uvrA and polA mutations in the presence or absence of exogenous metabolic activation. However, when TEA was mixed with sodium nitrite, mutations were induced in this system without exogenous metabolic activation; this activity was lost in the presence of exogenous metabolic activation.

TEA did not induce gene conversion in Saccharomyces cerevisiae in the presence or absence of exogenous metabolic activation in one study (TSCATS 1989A). In a single study, sex-linked recessive lethal mutations were not induced in Drosophila melanogaster by treatment with TEA either by diet or injection. Unscheduled DNA synthesis was not induced in rat primary hepatocytes exposed to TEA in two studies. TEA did not induce sister chromatid exchanges in Chinese hamster ovary cells in either the presence or absence of exogenous metabolic activation. Chromosomal aberrations were not induced in rat liver cells, Chinese hamster lung cells or Chinese hamster ovary cells by in-vitro exposure to TEA. It did not induce cell transformation in Syrian hamster embryo cells.





Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information on the test item regarding genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information,the test substance is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.