Registration Dossier

Administrative data

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 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (OECD TG 471, GLP); tested up to cytotoxic concentrations

- Mammalian cell gene mutation assay with Mouse Lymphoma (L5178Y) cells (met. act.: with and without) (OECD Guideline 476, GLP); tested up to cytotoxic concentrations; read-across from source substance MDEA-Esterquat C16-18 and C18 unsatd.

- In vitro mammalian chromosome aberration test with Chinese Hamster Lung (CHL) cells (met. act.: with and without) (OECD Guideline 473, GLP); cytotoxicity: no; read-across from source substance MDEA-Esterquat C16-18 and C18 unsatd.

- in vivo bone marrow micronucleus assay in NMRI BR mouse (OECD guideline 474, GLP); tested up to maximum recommended dose of 2000 mg/kg bw, intraperitoneal; no toxic effects; read-across from source substance MDEA-Esterquat C16-18 and C18 unsatd.

Link to relevant study records

Referenceopen allclose all

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: Guideline study, GLP
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his (Salmonella strains), trp (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
dose range finding test/first experiment: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate
second experiment: 10, 33, 100, 333 and 1000 µg/plate in Salmonella strains; 100, 333, 1000, 3330 and 5000 µg/plate in E. coli strain

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; (without metabolic activation)
Positive controls:
yes
Positive control substance:
furylfuramide
other: 2-aminoanthracene; (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48±4 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: eduction of the bacterial background lawn, increase in the size of the microcolonies, reduction of the revertant colonies
Evaluation criteria:
The test is considered acceptable if it meets the following criteria:
- The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
- The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
- The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
- In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 3330 and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: all values were within the range of historical controls
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

MDIPA-Esterquat C16-18 and C18 unsatd. was not mutagenic in this bacterial reverse mutation assay when tested up to cytotoxic concentrations.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted July 21, 1997) and EU method B.13/14 (31 May 2008), strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and E. coli  WP2 were exposed to MDIPA-Esterquat C16-18 and C18 unsatd. (100% a.i.) in ethanol at concentrations of 0, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the first experiment and 0, 10, 33, 100, 333 and 1000 µg/plate (Salmonella strains) and 0, 100, 333, 1000, 3330 and 5000 µg/plate (E. coli strain) in the second experiment in the presence and absence of mammalian metabolic activation (S9 mix).  

MDIPA-Esterquat C16-18 and C18 unsatd. was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2008-06-09 to 2008-07-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Treatment medium= McCoy's 5A medium supplemented with 10 % FBS, 100 units penicillin and 100 µg streptomycin/mL, 2 mM L-glutamine and 2.5 µg/mL amphotericin B (activated assay were conducted with medium without serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; The freeze lot of cells was tested using the Hoechst staining procedure.
- Periodically checked for karyotype stability: In order to assure the karyotypic stability of the cell line, cells were not used beyond passage 20.
- Periodically "cleansed" against high spontaneous background: N/A
- Other: The cells were obtained on November 19, 2003 from American Type Culture Colletion, Manassas, VA.
Additional strain / cell type characteristics:
other: CHO-K1 cells (repository number CCL 61)-averge cell cycle time of 10-14 hours with a modal chromosome number of 20
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Without S9 activation
1. 4 hour treatment- 25, 50, 100, 200, 225, 250 and 275 µg/mL
2. 20 hour treatment- 25, 50, 100, 125, 150, 175 and 200 µg/mL
With S9 activation
1. 4 hour treatment- 25, 50, 100, 200, 225, 250, 275 and 300 µg/mL
From the definitive chromosome aberration assay 2 x 12 mL aliquots of the vehicle and the lowest dosing preparation and 2 x 3 mL aliquots of the highest dosing preparation were collected for concentration analysis.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol (Lot No. B0514580)
- Justification for choice of solvent/vehicle: Ethanol was chosen as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a workable suspension in ethanol at a maximum concentration of 75 mg/mL in the solubility test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: (Without metabolic activation)
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: (With metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: N/A
- Exposure duration: 4 or 20 hours (without S9); 4 hours (with S9)
- Expression time (cells in growth medium): No expression time (20 hour treatment); 16 hours (4 hour treatments)
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours after the initiation of treatment (2 hours before harvest, Colcemid was added)
- Other: While there was not an expression time for the 20 hour treatment group, the flasks with visible precipitation were washed with 5 ml CMF-PBS prior to adding Colcemid to avoid precipitation interference with cell counts. Thus the treatment time for the precipitating dose levels was 18 hour instead of 20 hours.


SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5 % Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 metaphase spreads were analyzed per group (100 per replicate)


DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition- A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test substance precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. Cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control using the population doubling method.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Evaluation of Metaphase Cells-Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 +/- 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. To ensure a stable chromosome number in the cells used in the study prior to scoring, 25 metaphases were scored for chromosome number. A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid-type aberrations included chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and unanalyzable or severely damaged cell (>/= 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. The mitotic index was recorded as the percentage of cells in mitosis per 500 total cells counted. Polyploidy and endoreduplication was recorded as percentage per 100 metaphase cells counted.


OTHER: The pH of the highest concentration of dosing solution in treatment medium was measured using test tape. The selection of dose levels for analysis of chromosome aberrations in CHO cells was based upon toxicity of the test substance. The highest dose level selected for evaluation was the dose which induced at least 50 % toxicity, as measured by cell growth inhibition, relative to the solvent control, with a sufficient number of scorable metaphase cells. Two additional lower dose levels were included in the evaluation. At the initiation of treatment, two baseline control flasks from each exposure group were trypsinized and counted for cell number and cell viability, using Coulter counter and trypan blue dye exclusion. This baseline viable cell number was used to evaluate cell growth of the solvent controls during the course of the treatment and recovery periods.
Evaluation criteria:
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test substance was considered to induce a positive response when a statistically significant dose-related increase was observed in the number of aberrations in the test substance treated groups when compared to the control (p
Statistics:
In the preliminary and concurrent cytotoxicity studies, the cytotoxic effects of treatment were expressed as percent cell growth inhibition relative to the solvent treated control. In the chromosome aberration assay, the number and types of aberrations found were presented for each treatment flask. Duplicate treatment flasks were compared using the Fisher's exact test and, if not statistically different from each other, they were combined for treatment group comparisons. The percentage of damaged cells (numerical and structural) in the total population of cells examined as calculated for each group. Gaps were presented in the data but not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. For treatment groups where analyzable cells were scored, a "+" notation was made by the average aberrations per cell number to indicate that the value was a minimum. Pairwise comparison of the percent aberrant cells for each treatment to that of the solvent control was made using the Fisher's exact test. The p-values of the concentrations were adjusted to take into account multiple dose comparison (Bonferroni adjustment). The average number of aberrations per cells was reported but no statistical analysis was applied. The Cochran-Armitage trend test was performed between the solvent and treatment groups for each treatment condition and harvest time to test for dose responsiveness.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
N/A
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
N/A
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 1. Range-finding assay- the pH of the highest concentration of test substance (886 µg/mL) in treatment medium was approximately 7.4. 2. Chromosome aberration assay-The pH of the highest concentration of the test substance in treatment medium was approximately 7.4.
- Effects of osmolality: Range-finding assay- The osmolality in treatment medium of the highest concentration tested, 886 µg/mL, was 301 mmol/kg. The osmolality in treatment medium of the lowest precipitating concentration, 88.6 ug/ml, was 301 mmol/kg. The osmolality in treatment medium f the highest soluble concentration, 26.58 µg/mL, was 299 mmol/kg. The osmolality of the solvent (ethanol) in treatment medium was 301 mmol/kg. The osmolality of the test substance concentrations in treatment medium were acceptable because they did not exceed the osmolality by more than 20 %.
- Evaporation from medium: N/A
- Water solubility: N/A
- Precipitation: 1. Range-finding assay-The test substance formed a workable suspension in ethanol at 88.6 mg/mL and concentrations /= 88.6 µg/mL while dose levels /= 88.6 µg/mL) in all three treatment groups. 2. Chromosome aberration assay- The test substance was soluble in ethanol at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels >/= 100 µg/mL while does levels /= 50 µg/mL while dose level 25 µg/mL was soluble in treatment medium in all three treatment groups. Also, at the conclusion of the treatment period, no visible increase in floating cells were present at any dose level and the treatment medium contained particles of different sizes (some attached to the monolayer at dose levels >/= 50 µg/mL) in all three treatment groups.

- Other confounding effects: N/A


RANGE-FINDING/SCREENING STUDIES: The toxicity test was performed for the purpose of selecting concentrations for the chromosome aberration assay and consisted of assessing the test substance effect on cell growth, relative to the solvent control. At the initiation of treatment, two baseline control flasks from each exposure group were trypsinized and counted for cell number and cell viability, using Coulter counter and trypan blue dye exclusion. This baseline viable cell number was used to evaluate cell growth of the solvent controls during the course of the treatment and recovery periods.
CHO cells were seeded for each treatment condition at approximately 3-5 x 10^5 cells/25 cm² flask. Flasks were incubated at 37 +/- °C in a humidified atmosphere of 5 +/- % CO2 in air for 16-24 hours. Treatment was carried out by refeeding flasks with 5 ml complete medium (McCoy’s 5 A medium supplemented with 10 % FBS, 100 units penicillin and 100 µg streptomycin/mL, 2 mM L-glutamine and 2.5 µg/mL amphotericin B) for the non-activated study or S9 reaction mixture (4 mL complete medium plus 1 mL of S9 mix) for the S9 activated study, to which was added 50 µL dosing preparation of test substance in solvent or solvent alone. The osmolality in treatment medium of the solvent, the highest test substance concentration, the lowest precipitating test substance concentration and the highest soluble test substance concentration was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.
The cells were treated (0.0886 to 886 µg/mL) for 4 or 20 hours without S9 or for 4 hours with S9. Immediately after the completion of the non-activated 20-hour treatment, or 16 hours after the completion of the 4-hour treatments, the cells were harvested by trypsinization.
Trypan blue dye exclusion was used to determine cell viability and the cells were counted using a Coulter Counter to determine cell number, as a percentage, relative to the solvent control, for each exposure group. The cell growth in the treatment group relative to solvent control (population doubling), was calculated based on the following formula:
[(mean viable cells in test substance group-predosing cell count)/(mean viable cells in solvent control group-predosing cell count)x100]
Substantial toxicity (at least 50 % reduction in cell growth, relative to the solvent control), was observed at concentrations >/= 265.8 µg/mL in all three treatment groups




COMPARISON WITH HISTORICAL CONTROL DATA: All solvent and positive control values were within historical control value range.


ADDITIONAL INFORMATION ON CYTOTOXICITY and GENOTOXICITY:
1. 4 hour treatment in the absence of S9 activation- toxicity was 62 % at 200 ug/ml, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest concentration evaluated for chromosome aberrations, 200 ug/ml, was 17 % reduced relative to the solvent control. The concentrations selected for microscopic analysis were 50, 100, and 200 ug/ml. The percentage of cells with structural and numerical aberrations in the test substance treated group was not significantly increased relative to the solvent control, regardless of concentration (p> 0.05, Fisher's exact test with Bonferroni adjustment). The percentage of structurally damaged cells in the positive control group (18.5 %) was statistically significant.
2. 4 hour treatment in presence of S9 activation- Toxicity was 66 % at 225 ug/ml, the highest test substance concentration evaluated for chromosome aberrations. The mitotic index at the highest concentration evaluated for chromosome aberrations, 225 ug/ml, was 23 % reduced relative to the solvent control. The concentrations selected for microscopic analysis were 100, 200 and 225 ug/ml. The percentage of cells with structural and numerical aberrations in the test substance treated group was not significantly increased above that of the solvent control, regardless of concentration (p>0.05, Fisher's exact test with Bonferroni adjustment). The percentage of structurally damaged cells in the positive control treatment group (31.0 %) was statistically significant.
3. 20 hour treatment in the absence of S9-Toxicity was 69 % at 125 ug/ml, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest concentration evaluated for chromosome aberrations, 125 ug/ml, was 16 % reduced relative to the solvent control. The concentrations selected for microscopic analysis were 50, 100, and 125 ug/ml. The percentage of cells with structural and numerical aberrations in the test substance treated group was not significantly increased above that of the solvent control, regardless of concentration (p>0.05, Fisher's Exact test with Bonferroni adjustment). The percentage of structural damaged cells in the positive control treatment group (18.5 %) was statistically significant.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: N/A

Dose Level Analysis- The results of the analysis indicated that the analyzed samples (5 and 30 mg/ml) were 107.6 % and 97.6 % of their respective nominal concentrations. No test substance was detected in the vehicle control sample. This indicated that the dose formulations were accurately prepared and were acceptable for use in the study. Since the analyzed sample was within the protocol specified range of +/- 10 % of target, the dose formulations were considered stable for the purpose of the study.

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The objective of the study was to evaluate the potential of MDEA-Esterquat C16-18 and C18 unsatd. to induce structural and numerical chromosome aberrations in Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation. Based on the findings of the study, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the presence and absence of S9 in Chinese hamster ovary (CHO) cells.
Executive summary:

The objective of the study was to evaluate the potential of MDEA-Esterquat C16-18 and C18 unsatd. to induce structural and numerical chromosome aberrations in Chinese hamster ovary cells (CHO). Cytotoxicity in the preliminary and definitive chromosome aberration assays was determined by population doubling method. The solvent of choice was ethanol based on the solubility of the test substance and compatibility with the target cells. The test substance formed a workable suspension in ethanol at a maximum concentration of 75 mg/ml in the solubility test. The preliminary toxicity assay and chromosome aberration assay consisted of a 4 and 20 hour treatment period in the absence of metabolic activation and a 4 hour treatment in the presence of activation.  Cultures were harvested 20 hours after initiation of treatment. Positive (mitomycin C-non activated assay, Cyclophosphamide-activated assay) and vehicle controls were included for the chromosome aberration assay.

In the preliminary toxicity assay, the maximum concentration tested was 886 µg/mL. The test article formed a workable suspension in ethanol at 88.6 mg/mL and concentrations </= 26.58 mg/mL were soluble in ethanol. Visible precipitate was observed in treatment medium at dose levels >/= 88.6 µg/mL while dose levels </= 26.58 µg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period. At the conclusion of the treatment period, no visible increase in floating cells were present at any dose level and the treatment medium contained particles of different sizes (some attached to the monolayer at dose levels >/= 88.6 µg/mL) in all three treatment groups.

Substantial toxicity (at least 50 % reduction in cell growth, relative to the solvent control), was observed at concentrations >/= 265.8 µg/ml in all three treatment groups.  Based on these results the following concentrations were tested in the chromosome aberration assays:

Without S9 activation

1. 4 hour treatment- 25, 50, 100, 200, 225, 250 and 275 µg/mL (50, 10, and 200 µg/mL were analyzed for chromosome aberrations)

2. 20 hour treatment- 25, 50, 100, 125, 150, 175 and 200 µg/mL (50, 100, and 125 µg/mL were analyzed for chromosome aberrations)

With S9 activation

1. 4 hour treatment- 25, 50, 100, 200, 225, 250, 275 and 300 µg/mL (100, 200 and 225 µg/mL were analyzed for chromosome aberrations)

In the chromosome aberration assay, the test substance was soluble in ethanol at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels >/= 100 µg/mL while dose levels </= 50 µg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels >/= 50 µg/mL while doe level 25 µg/mL was soluble in treatment medium in all three treatment groups. Also, at the conclusion of the treatment period, no visible increase in floating cells were present at any dose level and the treatment medium contained particles of different sizes (some attached to the monolayer at dose levels >/= 50 µg/mL) in all three treatment groups.

Substantial toxicity (at least 50 % reduction in cell growth, relative to the solvent control), was observed at dose levels >/= 200 µg/mL in the non-activated 4 -hour treatment group, at dose levels >/= 225 µg/mL in the S9 activated 4 -hour exposure group, and at dose levels >/= 125 µg/mL in the non-activated 20 -hour treatment group. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50 % reduction in cell growth relative to solvent control) in all three treatment groups. Two lower doses were also selected for microscopic evaluation. The percentage of cells with structural or numerical aberrations n the test substance treated groups was not significantly increased above that of the solvent control at any concentration (p>0.05, Fisher's exact test with Bonferroni adjustment). Positive controls induced significant increases in chromosome aberrations for all assays.

The results of the assay are summarized in the following table:

Treatment Time (hours) Recovery Time (hours) Harvest Time (hours) S9 Toxicity at highest dose scored (µg/mL) Mitotic Inex Reduction LED for Structural Aberrations (µg/mL) LED for Numerical Aberrations (µg/mL)
4 16 20 - 62 % at 200 17% None None
20 0 20 - 69 % at 125 16% None None
4 16 20 + 66 % at 225 23% None None

Toxicity at highest dose scored in table= cell growth inhibition, relative to the solvent control

Mitotic Index Reduction in table=relative to solvent at high dose evaluated for chromsome aberrations

LED in table- Lowest Effective Dose

Based on the findings of the study, the test substance was concluded to be negative for the induction of structural and numerical chromsome aberrations in both the presence and absence of S9 in Chinese hamster ovary (CHO) cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1996-06-03 to 1996-07-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: P0P Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures-found to be free of contamination.
- Periodically checked for karyotype stability:no
- Periodically "cleansed" against high spontaneous background: yes. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK +/- cells by culturing in a restrictive medium.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Assay: 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 36 µg/mL. (presence and absence of S9-activation)
Mutagenesis Assay: 22.5, 25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (non-activated system) and 25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (S9-activated system).
Confirmatory Mutagenesis Assay: 5, 10, 20, 35, 60, 75, 90, 100, 110, 120 and 130 µg/mL (non-activated system) and 50, 60, 75, 90, 100, 110, 120, 130, 140, and 150 µg/mL (S9-activated system).
3rd Mutagenesis Assay: 35, 50, 60, 75, 90, 100, 110, 120, 130, 140 and 150 µg/mL (non-activated system) and 110, 120, 130, 140, 150, 200, 300, 400, 500, and 550 µg/mL (S9-activated).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone (CAS 67-64-1). The dosing solutions were adjusted to the acitve content of the test substance. Aliquots of dosing solution preparations were returned to the Sponsor for chemical analysis.
- Justification for choice of solvent/vehicle: acetone was chosen based on the Sponsor's request and compatibility with the target cells. The test substance was workable in acetone at 1.0 mg/mL. Concentrations of greater than or equal to 1.0 mg/mL were delivered to the test system as suspensions.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: (with metabolic activation)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: (without metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, combining 6 x 10 E6 L 5178Y/TK+/- cells and 100 µL dosing solution of test or control article in solvent or solvent alone in a total volume of 10 mL F0P medium or S9 activation mixture.

DURATION
- Preincubation period: Not applicable
- Exposure duration: 4 hours with mechaniclal mixing
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): TFT Medium (Trifluorthymidine)

NUMBER OF REPLICATIONS: 3 petri dishes for each sample/ 3 indipendent experiments

NUMBER OF CELLS EVALUATED: 1x10E6/plate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Not available.
- Determination of endoreplication: Not available.
Evaluation criteria:
- Cytotoxic effects: of each treatment condition were expressed relative to the solvent-treated control for suspension growth over 2 days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection).
- Mutant frequency: (number of mutants per 10E6 surviving cells) was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding V.C. plates and multiplying by the dilution factor (2x10E-4).
- Induced mutant frequency: calculated by subtracting the average mutant frequency of the solvent controls from the mutant frequency of the test substance treated cultures. Due to the use of non-rounded numbers by the computer system in calculations, the induced mutant frequencies presented in the data tables may indicate a slight discrepancy (typically by a value of 1) from the difference between the average solvent control mutant frequency (a rounded number) and the mutant frequency of the test substance treated cultures (rounded numbers).

-Positive: a result was considered positive if there was a positive dose response and one or more of the three highest doses in the 10% or greater total growth range exhibited a mutant frequency which was greater than or equal to 100 mutants per 10E6 clonable cells over the background level.
-Equivocal: a result was considered equivocal if the mutant frequency in treated cultures was between 55 and 100 mutants per 10E6 clonable cells over background level.
-Negative: a result was considered to be negative if the test articles which produced fewer than 55 induced mutants per 10E6 clonable cells at dose levels with greater than or equal to 10 % total growth.

Statistics:
Not applicable
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not available.
- Effects of osmolality: Preliminary Toxicity Assay-osmolality of the solvent control was 202 mmol/kg and the osmolality of the top dose, 36 µg/ml, was 323 mmol/kg. Osmolality of higher concentrations not measured; pH not measured.
- Evaporation from medium: not applicable.
- Water solubility: not available.
- Precipitation: Preliminary Toxicity Assay--treatment medium was cloudy but with no visible precipitate at a concentration of 36 µg/ml. Initial Mutagenesis Assay--treatment medium was cloudy but with no visible precipitate at concentrations of greater than or equal to 15 µg/ml. Confirmatory Mutagenesis Assay--treatment medium was cloudy but with no visible precipitate at concentrations of greater than or equal to 60 µg/ml. Third mutagenesis Assay-treatment medium was cloudy but with no visible precipitate at concentrations of greater than or equal to 60 µg/ml. The highest achievable dose was 550 µg/ml. No justification given. Might be precipitation.


RANGE-FINDING/SCREENING STUDIES: not available.


COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data for solvent and positive controls were given. Solvent controls of second test with metabolic activation were considered to be unacceptable due to high mutant frequencies of 136 and 167 induced mutants, corresponding historical data range of mean mutation frequency is 35,3 – 50,3 (1993 - 1995).


ADDITIONAL INFORMATION ON CYTOTOXICITY: not available.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

PRELIMINARY TOXICITY ASSAY: The maximum dose tested in the preliminary toxicity assay was 36 µg/mL. Concentrations of less than or equal to 10 µg/mL were soluble in treatment medium. Suspension growth relative to the solvent controls was 78 % at 36 µg/ml without activation and 92 % at 36 µg/ml with S9 activation. Based on the results of the toxicity test and the Sponsor's request, the doses chosen for the initital mutagenesis assay ranged from 1.0 to 75 µg/mL for both the non-activated and S9 -activated cultures.

INITIAL MUTAGENESIS ASSAY: In the non-activated system, cultures treated with test substance concentrations were cloned and produced a range in suspension growth of 28-81 %. In the S9 -activated system cultures treated with test substance concentrations were cloned and produced a range in suspension growth of 87-95 %. Concentrations of less than or equal to 10 µg/mL were soluble in treatment medium.

No treated cultures exhibited greater than or equal to 100 induced mutants per 10E6 clonable cells over the background level; three S9-activated cultures exhibited between 55 and 99 induced mutants per 10E6 clonable cells over the background level (40,60, and 75 µg/mL).

The total growths ranged from 23-76 % for the non-activated cultures at concentrations of 22.5-75 µg/mL and 70 -92 % for the S9 -activated cultures at concentrations of 25 -75 µg/mL. At the sponsor's request, the dose levels chosen for the confirmatory assay ranged from 1.0-150 µg/mL.

CONFIRMATORY MUTAGENESIS ASSAY: in the non-activated system, cultures treated with test substance concentrations of 5 to 120 µg/mL were cloned and produced a range in suspension growth of 17 -105 %. In the S9 -activated system cultures treated with test substance concentrations of 50 - 150 µg/mL were cloned and produced a range in suspension growth of 81 -95 %. Treatment medium was cloudy but with no visible precipitate at concentrations of  ≥ 60 µg/mL. Concentrations of less than or equal to 50 µg/mL were soluble in treatment medium.

No treated cultures exhibited greater than or equal to 100 induced mutants per 10E6 clonable cells over the background level. A dose-response trend was not observed in the non-activated or S9 -activated systems. The total growths ranged from 13 -90 % for the non-activated cultures at concentrations of 5 -120 µg/mL and 72 -103 % for the S9 -activated cultures. The solvent controls for the S9 -activated portion were unacceptable due to high mutant frequencies; however, the data are included in the report for completeness, along with the data from a repeat of the assay with S9 activation.

THIRD MUTAGENESIS ASSAY: performed since no cultures exhibited < 20 % total relative growth in the initial mutagenesis assay (despite the use of test substance concentrations that were cloudy in treatment medium) and the solvent controls for the S9 -activated portion were unacceptable in the confirmatory mutagenesis assay. At the Sponsor's request, an attempt was made to achieve a high dose of 1000 µg/mL in the S9 portion. The highest achievable dose was 550 µg/mL. The doses used in the third mutagenesis assay ranged from 35 -140 µg/mL without activation and from 110 -550 µg/mL with S9 activation. In the non-activated system, cultures treated with test substance concentrations were cloned and produced a range in suspension growth of 10 -66 %. In the S9 -activated system cultures treated with test substance concentrations were cloned and produced in a range suspension growth of 44 -78 %. Treatment medium was cloudy but with no visible precipitate at concentrations of ≥ 60 µg/mL. Concentrations of less than or equal to 50 µg/mL were soluble in treatment medium.

No treated cultures with greater than or equal to 10% total growth exhibited greater than or equal to 100 induced mutants per 10E6 clonable cells over the background level, one S9 -activated culture (300 µg/ml) exhibited 55 induced mutants per 10E6 clonable cells over the background level. A dose-response trend was not observed in the non-activated or S9 activated system. The total growths ranged from 5 – 40 % for the non –activated cultures at concentrations of 35 – 140 µg/mL and 20 – 68 % for the S9 –activated cultures at concentrations of 110 – 550 µg/mL.

For the 3 mutagenesis assays the TFT colonies for the positive and solvent control cultures were sized according to diameter over a range from approx. 0.2 to 1.1 mm.

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The test substance Reaction products of C16-18/C18 unsaturated fatty acid with methyl diethanolamine, MeCl quaternized was concluded to be negative with and without activation in the L5178Y/TK Mouse Lymphoma Mutagenesis Assay.
Executive summary:

In a mammalian cell gene mutation assay thymidine kinase locus comparable to OECD guideline 476, L5178Y mouse lymphoma cells cultured in vitrowere exposed to MDEA-Esterquat C16-18 and C18 unsatd. at the following concentrations in the presence and absence of mammalian metabolic activation (S9- mix of Arochlor 1254 induced rat liver).

Preliminary Toxicity Assay: 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 36 µg/mL (presence and absence of S9-activation)

Mutagenesis Assay: 22.5, 25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (non-activated) and

25, 27.5, 30, 32.5, 35, 40, 50, 60, and 75 µg/mL (S9 -activated).

Confirmatory Mutagenesis Assay: 5, 10, 20, 35, 60, 75, 90, 100, 110, 120 and 130 µg/mL (non-activated) and 50, 60, 75, 90, 100, 110, 120, 130, 140, and 150 µg/mL (S9-activated).

3rd Mutagenesis Assay: 35, 50, 60, 75, 90, 100, 110, 120, 130, 140 and 150 µg/mL (non-activated system) and 110, 120, 130, 140, 150, 200, 300, 400, 500, and 550 µg/mL (S9-activated).

Acetone was selected as solvent and was workable at concentrations of 1.0 mg/mL. Higher concentrations were delivered to the test system as suspensions.

MDEA-Esterquat C16-18 and C18 unsatd. was tested up to cytotoxic concentrations of ≥ 110 µg/mL without metabolic activation in the second and third assay. With metabolic activation in the third test a relative total growth of 20 % was observed at a concentration of 500 µg/ml (at 550 µg/mL the relative total growth was 31 %). According to the authors 550 µg/mL was the highest achievable concentration.

No treated cultures without metabolic activation with ≥ 10 % total growth exhibited ≥  55 induced mutants per 10E6 clonable cells over background level. No treated cultures with metabolic activation with ≥ 10 % total growth exhibited ≥ 100 induced mutants per 10E6 clonable cells over background level. Whereas 4 cultures with metabolic activation with ≥ 10 % total growth show induced mutant frequency of ≥ 55 induced mutants per 10E6 clonable cells over background at a concentration of 300 µg/mL in the third. assay and  40, 60 and 75 µg/mL in the first assay, respectively. The positive controls didinduce the appropriate response. 

The results of the L 5178Y/ TK Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study MDEA-Esterquat C16-18 and C18 unsatd. did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative under the conditions of this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-09-28 to 2012-12-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: NMRI BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males 33.9 ± 1.9 g, females 28.7 ± 2.0 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in groups (maximum 5 animals per sex per cage) in Macrolon cages (type MIII height: 15 cm) containing sterilised sawdust as bedding material
- Diet (e.g. ad libitum): pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 21.5°C
- Humidity (%): 38 - 66%
- Air changes (per hr): app. 15/h
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not given
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
prepared on the day of administration
- dosing volume: 10 mL/kg bw
Duration of treatment / exposure:
24 h
Frequency of treatment:
1x
Post exposure period:
24 h (+48 h for 2000 mg/kg bw dose group)
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:

No. of animals per sex per dose:
5 (main test)
3 (pretest)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on results of a dose range finding test

DETAILS OF SLIDE PREPARATION:
- bone marrow was flushed from femurs with approximately 2 mL of fetal calf serum
- cell suspension was collected and centrifuged at 216 g for 5 min
- supernatant was removed with a Pasteur pipette leaving one drop of serum on the pellet, cells were mixed with the remaining serum
- a drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol and cleaned with a tissue
- the drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension; the preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight
- two slides were prepared per animal
- staining with Giemsa
METHOD OF ANALYSIS:
- slides were scored at a magnification of 1000 x
- the number of micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%)
- the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time
- micronuclei were only counted in polychromatic erythrocytes
- averages and standard deviations were calculated
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
- The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
- The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
- The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: hunched posture, rough coat, lethargy

RESULTS OF DEFINITIVE STUDY
- animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality
- 1000 and 2000 mg/kg bw: hunched posture, rough coat, lethargy
- 500 mg/kg bw: hunched posture, rough coat

- Induction of micronuclei (for Micronucleus assay): no increase in the mean frequency of micronucleated polychromatic erythrocytes was observed
- Ratio of PCE/NCE (for Micronucleus assay): no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis
- Appropriateness of dose levels and route:
tested up to maximum recommended dose in accordance with current regulatory guidelines
route of administration (intraperinoneal) was chosen to maximize the chance of the test substance reaching the target tissue

Treatment mg/kg bw

Sampling time [h]

Number of micronucleated polychromatic erythrocytes (mean ± SD)

Ratio polychromatic/ normochromatic erythrocytes (mean ± SD)

males

0 (vehicle)

24

2.2 ±1.3

0.95 ±0.06

2000

24

2.0 ±0.7

0.68 ±0.31

2000

48

1.2 ±0.4

0.70 ±0.20

1000

24

2.0 ±1.4

0.69 ±0.15

500

24

2.2 ±1.3

0.81 ±0.13

Cyclophosphamide, 40 mg/kg bw

48

25.4 ± 4.2*

0.47 ±0.22

females

0 (vehicle)

24

1.8 ±0.8

0.96 ±0.06

2000

24

2.8 ±1.1

0.78 ±0.22

2000

48

0.4 ±0.5

0.79 ±0.14

1000

24

2.8 ±1.1

0.84 ±0.10

500

24

1.4 ±0.9

0.81 ±0.15

Cyclophosphamide, 40 mg/kg bw

48

22.8 ± 5.0*

0.62 ±0.12

* Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Conclusions:
Interpretation of results (migrated information): negative
MDEA-Esterquat C16-18 and C18 unsatd. was evaluated for clastogenic potential in the NMRI BR mouse bone marrow micronucleus assay at doses of 0, 500, 1000 and 2000 mg/kg bw. Under the conditions of the study, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.
Executive summary:

In a NMRI BR mouse bone marrow micronucleus assay according to OECD guideline 474 (adopted July 21, 1997) and EU method B.12 (31 May 2008) 5 animals/sex/dose were treated by intraperitoneal injection with MDEA-Esterquat C16-18 and C18 unsatd. at doses of 0, 500, 1000 and 2000 mg/kg bw.  Bone marrow cells were harvested at 24 h for all dose levels and additionally at 48 h at 2000 mg/kg bw post-treatment.  The vehicle was corn oil.

Hunched posture, rough coat and lethargy were observed at 1000 and 2000 mg/kg bw; hunched posture and rough coat were also present at 500 mg/kg bw.

MDEA-Esterquat C16-18 and C18 unsatd. was tested up to the maximum recommended dose in accordance with current regulatory guidelines. No decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. However the route of exposure was chosen to maximise the chance of the test substance reaching the target tissue.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro tests

Reverse gene mutation assays in bacteria

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted July 21, 1997) and EU method B.13/14 (31 May 2008), strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and E. coli  WP2 were exposed to MDIPA-Esterquat C16-18 and C18 unsatd. (100% a.i.) in ethanolat concentrations of 0, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the first experiment and 0, 10, 33, 100, 333 and 1000 µg/plate (Salmonella strains) and 0, 100, 333, 1000, 3330 and 5000 µg/plate (E. coli strain) in the second experiment in the presence and absence of mammalian metabolic activation (S9 mix).  

MDIPA-Esterquat C16-18 and C18 unsatd. was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background.

 

Both structurally related source substances, MDIPA Esterquat C18 unsatd. and MDEA-Esterquat C16-18 and C18 unsatd. , were also negative for mutagenic potential in the bacterial reverse mutation assay:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to MDIPA Esterquat C18 unsatd. (100% a.i.) in the presence and absence of mammalian metabolic activation (rat liver S9 mix).

Under the conditions of the study, MDIPA Esterquat C18 unsatd. was negative for mutagenic potential when tested up to cytotoxic concentrations.

 

Also for the sourde substance MDEA-Esterquat C16-18 and C18 unsatd., a reverse bacterial gene mutation assay according to OECD Guideline 471 (February 1998) was negative up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (S9-mix) in S. Typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA (pKM101).

 

Mammalian cell gene mutation assay

Additional data are for the source substance MDEA-Esterquat C16-18 and C18 unsatd. is available from a mammalian cell gene mutation assay (thymidine kinase locus) comparable to OECD Guideline 476, performed with mouse lymphoma L 5178Y cells. Cells cultured in vitro were exposed to MDEA-Esterquat C16-18 and C18 unsatd. at concentration up to 150 µg/mL in the absence and up to 550 µg/mL in the presence of mammalian metabolic activation (S9- mix of Aroclor 1254 induced rat liver).

Substantial toxicity with a suspension growth of ≤ 50 % was observed at ≥ 50 µg/mL without metabolic activation and at ≥ 150 µg/mL with metabolic activation.

In the first experiment the concentration range did not cover cytotoxicity in the test with metabolic activation. Due to unacceptable high solvent controls of the test with metabolic activation in the confirmatory assay a third independent assay was performed.

No treated cultures with or without metabolic activation with ≥ 10 % total growth exhibited ≥ 100 induced mutants per 1E06 clonable cells over background level, the limit value for a positive response. No treated cultures without metabolic activation with ≥ 10 % total growth exhibited ≥ 55 to 99 induced mutants per 1E06 clonable cells over background level, the limit values for equivocal response.

Three cultures with metabolic activation with ≥ 10 % total growth showed induced mutant frequency of ≥ 55 induced mutants per 1E06 clonable cells over background at a concentrations of 40, 60 and 75 µg/mL in the first assay (63, 71 an 56 induced mutants per 1E06 clonable cells over background, respectively). A mutant number in the range of negative results was observed at the intermediate concentration of 50 µg/mL, (41 induced mutants per 1E06 clonable cells over background). A dose-response trend was not observed. 

In the third assay much higher concentrations from 110 to 550 µg/mL were tested, due to the missing cytotoxicity in the first assay. At the intermediate concentration of 300 µg/mL, a mutant number equivalent to the lower bound for equivocal results of 55 induced mutants per 1E06 clonable cells over background was observed. A dose-response trend was not observed.

The positive controls did induce the appropriate response. 

The results of the L 5178Y/ TK Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study the MDEA-Esterquat C16-18 and C18 unsatd. did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative.

 

Mammalian cell cytogenetics assay

In a mammalian cell cytogenetics assay, chromosome aberration test comparable to OECD Guideline 473 (21 July 1997), CHO cell cultures were exposed to the source substance MDEA-Esterquat C16-18 and C18 unsatd. (99.8 % a.i.), at concentrations of 25, 50, 100, 200, 225, 250 and 275µg/mL in the non-activated 4-hour treatment group and of 25, 50, 100, 125, 150, 175 and 200 µg/mL in the non-activated 20-hour treatment group. 

Aroclor 1254-induced rat liver S9-mix was used for metabolic activation at test substance concentrations of 25, 50, 100, 200, 225, 250, 275 and 300 µg/mL in the activated 4-hour treatment group.

Substantial toxicity (at least 50% reduction in cell growth, relative to the solvent control), was observed at dose levels ≥ 200μg/mL in the non-activated 4-hour treatment group, at dose levels ≥ 225μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 125μg/mL in the non-activated 20-hour treatment group.

Selection of doses for microscopic analysis was based on toxicity including the lowest dose with at least 50% reduction in cell growth relative to solvent control in all three treatment groups. The following doses were selected for microscopic evaluation:

non-activated 4-hour treatment group: 50, 100 and 200 μg/mL

S9-activated 4-hour exposure group: 100, 200 and 225 μg/mL

non-activated 20-hour treatment group: 50, 100 and 125 μg/mL

Positive controls induced the appropriate response.

MDEA-Esterquat C16-18 and C18 unsatd. was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the presence and absence of metabolic activation in Chinese hamster ovary (CHO) cells.

 

IN VIVO TEST

Mouse bone marrow micronucleus assay

In a NMRI BR mouse bone marrow micronucleus assay according to OECD guideline 474 (adopted July 21, 1997) and EU method B.12 (31 May 2008) 5 animals/sex/dose were treated by intraperitoneal injection with the source substance MDEA-Esterquat C16-18 and C18 unsatd. at doses of 0, 500, 1000 and 2000 mg/kg bw.  Bone marrow cells were harvested at 24 h for all dose levels and additionally at 48 h at 2000 mg/kg bw post-treatment.  The vehicle was corn oil.

Hunched posture, rough coat and lethargy were observed at 1000 and 2000 mg/kg bw; hunched posture and rough coat were also present at 500 mg/kg bw. MDEA-Esterquat C16-18 and C18 unsatd. was tested up to the maximum recommended dose in accordance with current regulatory guidelines. No decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. However the route of exposure was chosen to maximise the chance of the test substance reaching the target tissue. 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.

 

Based on the available reliable, relevant and adequate data, there was no evidence of genotoxicity for MDIPA-Esterquat C16-18 and C18 unsatd. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Endpoint specific justification for read-across

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across given in chapter 5 of the CSR and attached as pdf document to section 7 of the IUCLID file.

 

Structural similarity

a. Structural similarity and functional groups

The target substance, MDIPA-Esterquat C16-18 and C18 unsatd., consists of an amine backbone (MDIPA = Methyldiisopropanol amine) esterified with long chain fatty acids C16, C18 and C18 unsaturated. The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Methosulfate.

The first source substance, MDEA-Esterquat C16-18 and C18 unsatd., consists of an amine backbone (MDEA = Methyldiethanol amine) esterified with long chain fatty acids C16, C18 and C18 unsaturated. The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Chloride.

The second source substance, MDIPA-Esterquat C18 unsatd., consists of an amine backbone (MDIPA = Methyldiisopropanol amine) esterified with long chain fatty acids C18 unsaturated. The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Methosulfate.

The source and the target substances share structural similarities with common functional groups (quaternary amines), esters, and fatty acid chains with comparable length and degree of saturation. The amine backbones based on MDEA and MDIPA, respectively, differ only by one methyl group in both chains, all functional groups are identical.

 

b. Common breakdown products

The metabolism expected to occur is hydrolysis of the ester-bond by esterases. However, the rate of hydrolysis is assumed to be low. The fraction of metabolised molecules would result in free fatty acids and Dimethyl-DEA (DEA = Diethanolamine) and Dimethyl-DIPA (DIPA = Diisopropanolamine), respectively. The carboxylic acids are further degraded by the mitochondrial beta-oxidation process (for details see common text books on biochemistry). The fatty acids enter normal metabolic pathways and are therefore indistinguishable from fatty acids from other sources including diet. The quaternary ammonium ions are not expected to be further metabolised, but excreted unchanged via the urine.

 

c. Differences

The differences in fatty acid chain length (higher percentage of C16 in the source substance MDEA-Esterquat C16-18 and C18 unsatd. ) and degree of saturation (higher degree of unsaturation in the source substance MDIPA-Esterquat C18 unsatd.) may be relevant for local effects (e.g. irritation) but are not considered to be of relevance for genotoxicity.

Chloride is an essential nutrient and present in all organisms; excess chloride is renally excreted (see common textbooks on biology / biochemistry). Methyl sulphate is metabolised to Sulphate and Carbon dioxide, and these are excreted via the urine and released by the lungs, respectively. The anions Chloride and Methyl sulphate are not expected to have any influence on toxicity or reactivity.

The methyl side chain of Dimethyl-DIPA which is not present in Dimethyl-DEA is not expected to enhance reactivity, which is supported by a similar toxicological profile for the source and target substance as well as toxicokinetic data for DEA, MDEA, DIPA and MDIPA (for details see general justification for read-across).

 

Comparison of genotoxicity data

Genotoxicity data of the target and source substances

Table 1: Comparison of genotoxicity data of MDEA-Esterquat C16-18 and C18 unsatd. , MDIPA Esterquat C18 unsatd. and MDIPA-Esterquat C16-18 and C18 unsatd.

 

 

Source substances

Target substance

Endpoints

MDEA-Esterquat C16-18 and C18 unsatd.

MDIPA Esterquat C18 unsatd.

MDIPA Esterquat C16-18 and C18 unsatd.

Genotoxicity in vitro

OECD TG 471 (Ames test)
RL 1, GLP,
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 (met. act.: with and without)

 

negative for all strains (with and without metabolic activation)

 

Doses:

1ststudy: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate

2ndstudy: 15, 50, 150, 500, 1500 and 5000 µg/plate

 

cytotoxicity: no, but tested up to precipitating concentrations

 

non-mutagenic

 

OECD TG 471 (Ames test)
RL 1, GLP
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (met. act.: with and without)

 

negative for all strains (with and without metabolic activation)

 

Doses:

1ststudy: 0 (control), 62, 185, 556, 1667 and 5000 µg/plate

2ndstudy: 0 (control), 22, 67, 200, 600 and 1800 µg/plate

 

 

cytotoxicity: yes

 

non-mutagenic

 

 

OECD TG 471 (Ames test)
RL 1, GLP
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (met. act.: with and without)

 

negative for all strains (with and without metabolic activation)

 

Doses: 1ststudy: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate

2ndstudy: 10, 33, 100, 333 and 1000 µg/plate in Salmonella strains; 100, 333, 1000, 3330 and 5000 µg/plate in E. coli strain

 

cytotoxicity: yes

 

non-mutagenic

 

OECD TG 476 (Mammalian cell gene mutation, MLY), RL 2, GLP

negative for mouse lymphoma L5178Y cells (with and without metabolic activation)

cytotoxicity: yes

 

non-mutagenic

 

Read-across from the source substance

Read-across from the source substance

OECD TG 473 (In vitro mammalian chromosome aberration), RL 1, GLP

negative for Chinese Hamster Ovary (CHO) (with and without metabolic activation)

cytotoxicity: yes

 

negative

 

Read-across from the source substance

Read-across from the source substance

Genotoxicity in vivo

 

 

OECD TG 474, mouse, i.p. RL 1, GLP

Doses:0, 500, 1000, 2000 mg/kg bw

toxicity: no effects

 

negative

 

Read-across from the source substance
No data requirement for REACH

Read-across from the source substance
No data requirement for REACH

Sensitisation

 

OECD TG 406 (2 Buehler tests)
RL 2, GLP

3 HRIPT tests: negative

 

negative (not sensitizing)

OECD TG 406 (M&K test)
RL 1, GLP

 

 

negative (not sensitizing)

 

OECD TG 406 (M&K test)
RL 1, GLP

 

 

 

negative (not sensitizing)

 

 

The source substances MDEA-Esterquat C16-18 and C18 unsatd. and MDIPA Esterquat C18 unsatd., and the target substance showed the same negative results in the bacterial reverse mutation assay (not mutagenic up to cytotoxic concentrations). There was no evidence of induced mutant colonies over background for any substance. These results further justify the read-across approach by extrapolation from the source substances for the mammalian gene mutation assay and the mammalian chromosome assay as well as the mouse bone marrow micronuleus assay.

Certain endpoints such as skin sensitisation and genotoxicity are characterised by covalent binding as a rate determining step or MIE (molecular initiating event). The consistency across endpoints - both, source substances and target substance were not sensitising - also helps to increase confidence in the read-across approach especially when MIEs are common for example, skin sensitisation and genotoxicity are underpinned by electrophilicity.

In the available sub-chronic study there are no indications of non-specific organ damage or chronic inflammation.

There are differences in local effects: MDEA-Esterquat C16-18 and C18 unsatd. was not irritating to the eye or skin, whereas MDIPA Esterquat C18 unsatd. had irreversible effects on the eye (Cat. 1) and was irritating to the skin (Cat. 2) and the target substance MDIPA-Esterquat C16-18 and C18 unsatd. was irritating to eyes (Cat. 2) and skin (Cat. 2). However, these differences in irritation properties are of no relevance for genotoxicity.

Overall the available data are comparable for source and target substance, supporting the validity of the grouping approach.

 

Quality of the experimental data of the analogues:

The source chemical MDEA-Esterquat C16-18 and C18 unsatd. has been tested in reliable OECD TG 471, OECD TG 476 and OECD TG 473 test. All tests have been conducted according to GLP criteria.

The second source substance MDIPA-Esterquat C18 unsatd. has been tested in a reliable GLP-compliant OECD TG 471 study. Therefore this data have no uncertainties and can be used in an analogue approach.

For the target chemical MDIPA Esterquat C16-18 and C18 unsatd. a study according to OECD TG 471 is available, which was also conducted in compliance with GLP.

The available data from the source chemical are sufficiently reliable to justify the read-across approach.

 

Classification and labelling (Human Health)

The first source substance MDEA-Esterquat C16-18 and C18 unsatd. is not classified for any human health hazard, whereas the second source substance MDIPA Esterquat C18 unsatd. is classified for local effects (irreversible effects on the eye Category 1, irritating to the skin Category 2).

The target substance MDIPA Esterquat C16-18 and C18 unsatd. is classified for local effects (irritating to eye Category 2, irritating to the skin Category 2).

 

Conclusion

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles as demonstrated in detail in the general justification for read-across.

The negative results from the bacterial reverse mutation assay, which is available for the source substances and the target substance, justify this read-across approach.

Further support is given by the lacking skin sensitisation potential for both, source and target substances. The endpoint sensitisation is – similar to the endpoint genotoxicity – based on covalent binding of the substance itself or reactive metabolites to cellular macromolecules as rate determining step. The consistency across the endpoints increases the confidence in the conclusion that there is no concern for reactive metabolites. 

 

The negative outcome of the complete testing battery of in vitro and in vivo genotoxicity tests for MDEA-Esterquat C16-18 and C18 unsatd. is considered to be relevant also for the target MDIPA-Esterquat C16-18 and C18 unsatd. No classification for genotoxic properties is required.

Justification for classification or non-classification

Based on the available reliable, relevant and adequate data on MDIPA-Esterquat C16-18 and C18 unsatd. and the source substance MDEA-Esterquat C16-18 and C18 unsatd., MDIPA-Esterquat C16-18 and C18 unsatd. does not need to be classified as genotoxic according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008. No labelling is required.