Fatty acids, C18 (even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate quaternized

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
• they share structural similarities with common functional groups: One quaternised ethanolamine moiety, one to three, mainly two ester groups with a typical UVCB distribution with long-chain fatty acids of natural origin. The molecular structure is almost identical.
• they are manufactured from similar resp. identical precursors (triethanolamine, long-chain fatty acids, dimethyl sulphate) under similar conditions. Therefore common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident
• A constant pattern in the changing of the potency of the properties across the TEA-Esterquats by chain-length and the grade of esterification is not observed, because the fatty acid chain-length distribution is too narrow and similar and the distribution of mono-, di-, and tri-esters is identical. Some variation caused by variation in C=C double bonds may occur and will be discussed at the relevant endpoint.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See general justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See general justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See general justification for read-across attached to chapter 13 of this IUCLID file.

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

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 this study.

Reference 2

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
• they share structural similarities with common functional groups: One quaternised ethanolamine moiety, one to three, mainly two ester groups with a typical UVCB distribution with long-chain fatty acids of natural origin. The molecular structure is almost identical.
• they are manufactured from similar resp. identical precursors (triethanolamine, long-chain fatty acids, dimethyl sulphate) under similar conditions. Therefore common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident
• A constant pattern in the changing of the potency of the properties across the TEA-Esterquats by chain-length and the grade of esterification is not observed, because the fatty acid chain-length distribution is too narrow and similar and the distribution of mono-, di-, and tri-esters is identical. Some variation caused by variation in C=C double bonds may occur and will be discussed at the relevant endpoint.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See general justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See general justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See general justification for read-across attached to chapter 13 of this IUCLID file.

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Precipitation:
Main experiments:
Precipitation of the test item visible to the unaided eye was noted at 20 µg/mL and above in the first experiment without metabolic activation and at 93.8 µg/mL and above in the first experiment with metabolic activation. In the second experiment precipitation as described above occurred at 45.0 µg/mL and above without metabolic activation and at 50.0 µg/mL and above with metabolic activation.


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 3000 µg/mL limited by the solubility of the test item in THF. Test item concentrations between 23.4 µg/mL and 3000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative clon-ing efficiency below 50 % were noted at 23.4 µg/mL and above without metabolic activa-tion and 375 µg/mL and above with metabolic activation following 4 h treatment. Following 24 hours treatment without metabolic activation cytotoxic effects as described above were noted at 187.5 µg/mL and above.

The test medium of the pre-experiment was checked for precipitation at the end of each treatment period (4 or 24 hours) just prior to removal of the test item. Precipitation was noted at 187.5 µg/mL and above after 4 and 24 hours treatment without metabolic activa-tion and at 375 µg/mL and above after 4 hours treatment with metabolic activation.

There was no relevant shift of pH or osmolarity of the medium even at the maximum con-centration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range of the first experiment up into the precipitating concentration range. An additional larger step was used to include the maximum possible concentration level limited by the solubility properties of the test item. The toxic gradient did not follow a smooth and dose dependent course in the pre-experiment following 4h treatment without metabolic activation. Therefore, the experimental part 4h treatment without metabolic activation of the first main experiment was started with 8 concentrations in order to obtain at least 4 analysable concentrations. In experiment II a narrower spacing was used at high concentrations to cover the precipitating or toxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Controls within the range of historical data


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects as indicated by a relative cloning efficiency I of less than 50 % in both parallel cultures occurred at 10 µg/mL and above in experiment I without metabolic activation and at 187.5 µg/mL and above with metabolic activation. In the second experiment toxic effects as described above occurred at 180 µg/mL without metabolic activation. The cell density at the first subcultivation after treatment showed less severe cytotoxicity in the precipitating concentration range especially without metabolic activation. Even though the relative cloning efficiency 1 was zero or close to zero at 20 µg/mL in the first experiment and at 180 µg/mL in the second experiment without metabolic activation the corresponding values of the cell density remained within the acceptable range. Such a large difference in the onset of cytotoxicity is usually based on binding of the test item to proteins or lipids of cell membranes, serum albumin, or S9. The low-density cell cultures used to determine the cloning efficiency 1 are in such cases more sensitive towards toxic effects than the mass cell cultures used to determine mutagenicity. In the experimental parts with metabolic activation proteins and lipids of the S9 level out the influence of different cell densities. Protein binding effects are also the reason that higher concentrations are tolerated in the second experiment without metabolic activation in spite of the 24h treatment period. However, 10% foetal calf serum have to be added during 24h treatment compared to no foetal calf serum during 4h treatment. Protein binding test items are therefore, better tolerated during long term exposure.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the partially unsaturated TEA-Esterquat is considered to be non-mutagenic in this HPRT assay.

Reference 3

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
• they share structural similarities with common functional groups: One quaternised ethanolamine moiety, one to three, mainly two ester groups with a typical UVCB distribution with long-chain fatty acids of natural origin. The molecular structure is almost identical.
• they are manufactured from similar resp. identical precursors (triethanolamine, long-chain fatty acids, dimethyl sulphate) under similar conditions. Therefore common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident
• A constant pattern in the changing of the potency of the properties across the TEA-Esterquats by chain-length and the grade of esterification is not observed, because the fatty acid chain-length distribution is too narrow and similar and the distribution of mono-, di-, and tri-esters is identical. Some variation caused by variation in C=C double bonds may occur and will be discussed at the relevant endpoint.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See general justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See general justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See general justification for read-across attached to chapter 13 of this IUCLID file.

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:

It can be stated that under the experimental conditions reported, the test item did not induce structural 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.

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
• they share structural similarities with common functional groups: One quaternised ethanolamine moiety, one to three, mainly two ester groups with a typical UVCB distribution with long-chain fatty acids of natural origin. The molecular structure is almost identical.
• they are manufactured from similar resp. identical precursors (triethanolamine, long-chain fatty acids, dimethyl sulphate) under similar conditions. Therefore common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident
• A constant pattern in the changing of the potency of the properties across the TEA-Esterquats by chain-length and the grade of esterification is not observed, because the fatty acid chain-length distribution is too narrow and similar and the distribution of mono-, di-, and tri-esters is identical. Some variation caused by variation in C=C double bonds may occur and will be discussed at the relevant endpoint.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See general justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See general justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See general justification for read-across attached to chapter 13 of this IUCLID file.

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:

It can be stated that under the experimental conditions reported, the test item “partially unsaturated TEA-Esterquat” did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item “partially unsaturated TEA-Esterquat” is considered to be 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 C18 and C18 unsatd. TEA-Esterquat. 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.

 

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.

 

Similar results were obtained with the source substance MDEA-Esterquat C16-18 and C18 unsatd.: the substance did not show any genotoxic intrinsic properties in the Ames test, mouse lymphoma assay, chromosome aberration study and in vivo bone marrow micronucleus assay and is therefore considered to be nongenotoxic. These data are included into the dossier to demonstrate, that the substances have a similar toxicological profile.

 

Justification for classification or non-classification

Based on the available data, C18 and C18 unsatd. TEA-Esterquat does not need to be classified for mutagenicity according to regulation (EC) 1272/2008. Thus, no labelling is required.