TEA-Esterquat

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:
- Ames test with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (met. act.: with and without) (OECD TG 471, GLP); tested up to cytotoxic concentrations; read-across: partially unsaturated TEA-Esterquat
- Mammalian cell gene mutation assay (HPRT) in V79 cells (met. act.: with and without) (OECD Guideline 476, GLP); tested up to cytotoxic concentrations; read-across: partially unsaturated TEA-Esterquat
- In vitro mammalian chromosome aberration test with V79 cells (met. act.: with and without) (OECD Guideline 473, GLP); tested up to cytotoxic concentrations; read-across: partially unsaturated TEA-Esterquat

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

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: Histidin auxotroph

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (fraction of Spraque Dawley rat liver incuded with Aroclor 1254). Obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, USA.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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

Species / strain:

E. coli WP2 uvr A pKM 101

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

Conclusions:

negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (Minimal Essential Medium; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 (protein concentration 35.7 mg/mL and 28.9 mg/mL)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: water was chosen as solvent, test substance was suspended in deionised water immediately before treatment.
- Precipitation: was observed, corresponding concentrations were identified in the exposure concentration table under
"any other information on materials and methods".

RANGE-FINDING/SCREENING STUDIES:
In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000µg/mL were applied. Clear toxic effects were observed after 4 hrs treatment with 156.3 µg/mL and above in the absence of S9 mix and with 625µg/mL and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 78.1 µg/mL and above in the absence of S9 mix induced strong toxic effects.
In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 39.1 µg/mL and above in the absence and the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 288 mOsm, pH 7.2 versus 323 mOsm and pH 7.2 at 5000 µg/mL).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In this study, cytotoxicity was observed in all cytogenetic experiments. In the presence of S9 mix the cell numbers were strongly reduced after treatment with 600 µg/mL (32 % of control) at the 18 hrs preparation interval and with 500µg/mL (43 % if control) at the 28 hrs preparation interval.

MAIN TEST:
In experiment I and II, in the absence and the presence of S9 mix, no biological relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.8 % abberant cells, exclusive gaps) were close to the range of the solvent control values (0.0 – 1.5 % aberrant cells, exclusive gaps) and close to the range of our historical control data (0.0 – 4.0 % aberrant cells, exclusive gaps).
In the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). To prove this slightly increase value exceeding the upper border of our laboratory´s historical control data range an increased sample of 200 metaphase plates per culture was evaluated for cytogenetic damage.
However, the borderline value was confirmed. In addition, a dose related increase in the number of cells carrying structural chromosome aberrations (0.5 %, 2.5 % and 4.8 %) was observed after 4 hrs treatment at 28 hrs preparation interval in the presence of metabolic activation at the upper concentrations evaluated (37.5, 150.0 and 300.0 µg/mL) respectively.

A confirmatory experiment, designated experiment III, was performed to proof these observations. In the repeated experiment in the presence of S9 mix after 4 hrs treatment at prolonged 28 hrs preparation interval no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 – 3.0 % aberrant cells exclusive gaps) were close to the value of the solvent control (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data, (0.0 – 4.0 % aberrant cells, exclusive gaps).

Beside the aberration rates were dose related increased (1.0 %, 1.5 % and 3.0 %) in the concentration range evaluated (300 to 500 mg/mL), but the values were clearly within our laboratory´s control data range (0.0 – 4.0 % aberrant cells, exclusive gaps). Finally, the observations of experiment II in the presence of S9 mix (statistical significance, dose dependency, and borderline value) were not confirmed in experiment III and therefore they have to be regarded as biologically irrelevant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 – 2.5 %) as compared to the rates of the solvent controls (1.9 – 3.0 %).

EMS and CPA were used as positive controls and showed distinct increase in cells with structural chromosome aberrations.

Conclusions:

negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (Seromed)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

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:

not examined

Positive controls validity:

valid

Conclusions:

negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- in vivo bone marrow micronucleus assay in CFW 1 mouse (OECD guideline 474, GLP); tested up to 5000 mg/kg bw, oral: gavage; no toxic effects (slight reduction in the ratio of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow); read-across: partially unsaturated TEA-Esterquat

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: outbred albino mouse, strain CFW 1

Sex:

male/female

Route of administration:

oral: gavage

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Dose range finding study: 2 males, 2 females per group (3 groups)
Main study: 6 males, 6 females per group (5 groups)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

A slight reduction in the ration of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000, 5000 mg/kg bw
- Solubility: in bidest. water
- Application volume: 20 ml/kg bw
- No animal up to the highest dose died within the first three days or showed signs of severe morbidity. Slight piloerection was observed at all animals and all doses up to 24 hours after administration.
- Based on the results of this range-finding study 5000 mg/kg bw was selected as appropriate dose for the test.

RESULTS OF DEFINITIVE STUDY
- 5000 mg/kg bw was tested as the maximum dose in the main experiment at 24, 48 and 72 h. The volume administered was 20 ml/kg bw.
- Toxicity: No mortality was was noted after administration of the test item in any of the animals evaluated. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes was determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow.
- Signs of clinical examination: slight piloerection in all animals including positive and negative control.

RESULTS OF NEGATIVE CONTROL GROUP
- no mortality within 24 h
- was in the range of historical control data

RESULTS OF POSITIV CONTROL GROUP
- No mortality within 24 h
- Cyclophoshamide induced a statistically significant increase in the number of micronucleated cells in both sexes.

Conclusions:

negative in the Mammalian Erythrocyte Micronucleus Test

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No experimental data are available for the assessment of genetic toxicity of the target substance Esterification products of triglycerides C18 unsaturated with triethanolamine, dimethyl sulfate quaternized. However, reliable relevant data are available for the closely related source substance partially unsaturated TEA-Esterquat. A justification for read-across is attached to Iuclid section 13.

 

The full set of in vitro tests required by REACH Regulation Annexes VII and VIII is covered with the studies. There was no evidence of mutagenic or genotoxic intrinsic properties in any of the performed studies. Additional data from an in vivo mouse micronucleus Test are available, likewise showing no evidence to cause any chromosomal damage in the bone marrow of mice.

 

In vitro data

A reverse bacterial gene mutation assay (Ames-Test, plate incorporation assay) according to OECD Guideline 471(1997) with partially unsaturated TEA-Esterquat was negative up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (rat liver S9-mix 10 and 30 %) in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (pKM101).

Significant bacteriotoxic effects of varying severity were observed, depending on the test strain and the presence of metabolic activation. Generally, bacteriotoxicity was less pronounced in the presence of metabolic activation, especially at concentrations of 30 % S9-mix. Precipitation was observed at 1600 µg/plate and above. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation.

 

In a mammalian cell gene mutation assay (HPRT locus) according to OECD guideline 476, Chinese hamster lung fibroblasts (V79) cells cultured in vitro were exposed to partially unsaturated TEA-Esterquat (100 % a.i. of UVCB description) at concentrations up to 200 µg/mL in the absence and up to 1500 µg/mL in the presence of mammalian metabolic activation (rat liver S9). 

The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium and by cytotoxic effects. The assay was performed in two independent experiments, using two parallel cultures each. A treatment period of 4 hours was used for all experiments, with the exception of the second experiment with metabolic activation, were a 24 hour treatment period was selected for the cultures. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase of mutant colonies and, thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

The results of this HPRT assay indicate, that partially unsaturated TEA-Esterquat did not cause a positive response in the non-activated and S9-activated systems and was assessed to be negative under the conditions of this study.

 

In a mammalian cell cytogenicity assay according to OECD Guideline 473 1997, V79 cell cultures were exposed to partially unsaturated TEA-Esterquat at concentration ranges of 3.1 – 200 µg/mL without metabolic activation and 4.7 – 600 µg/mL in the presence of mammalian metabolic activation.

In experiment I and II, in the absence and the presence of S9 mix, no biological relevant increase in the number of cells carrying structural chromosome aberrations was observed. However, in the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). In addition, a dose related increase in the number of cells carrying structural chromosome aberrations was observed in experiment II with metabolic activation.

A confirmatory experiment III was performed to verify these observations. In the repeated experiment in the presence of S9 mix after

4 hrs treatment at a prolonged 28 hrs preparation interval no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. Although the aberration rates showed a dose related increase, the values were clearly within the historical control data range of the testing laboratory. Finally, the observations of experiment II in the presence of S9 mix were not confirmed in experiment III and therefore they have to be regarded as biologically insignificant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found.

In conclusion it can be stated that under the experimental conditions reported, the test item did not induce structural or numeric chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.

Supporting data were identified on Glycerides: Medium- and long-chain triacylglycerols (MLCT; containing mainly C18:1 and C18:2 fatty acid glycerides) did not induce gene mutations in 4 different Salmonella and one E. coli strain in a bacterial reverse mutation assay (plate incorporation) with and without metabolic activation. Short- and long-chain fatty acid triacylglycerols (SLCT; containing mainly C18:0 fatty acid glycerides) were negative in a chromosome aberration assay in CHO cells with and without metabolic activation as well as in a gene mutation study in mammalian cells (HPRT test) in CHO cells with and without metabolic activation.

 

In vivo data

In a mouse bone marrow micronucleus assay according to OECD guideline No. 474, 1983, 6 male and 6 female albino mice (CFW1) per group were treated by oral intubation with partially unsaturated TEA-Esterquat at a dose of 5000 mg/kg bw. Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.

There were no signs of toxicity as indicated by an enhanced mortality rate. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes were determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow. The partially unsaturated TEA-Esterquat was tested at an adequate dose, based on the results of the range-finding test. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

 

There are no data gaps for the endpoint genetic toxicity. No human data are available. However, there is no reason to believe that these results from rat and rabbits would not be applicable to humans.

Justification for classification or non-classification

Based on the available data, Esterification products of triglycerides C18 unsaturated with triethanolamine, dimethyl sulfate quaternized does not need to be classified for mutagenicity according to regulation (EC) 1272/2008. Thus, no labelling is required.