Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-09 to 2008-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides

Cas Number:

1079184-43-2

Molecular formula:

n.a. (UVCB)

IUPAC Name:

Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): Reaction products of C16-18 (C18 unsatturated) fatty acid with mehtyl diethanolamine, MeCl quaternized
- Diethylester dimethyl ammonium chloride
- Physical state: White powder

Method

Target gene:

N/A

Species / strain

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.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

SUMMARY OF CYTOGENETIC ANALYSIS

Treatment [µg/mL]	Treatment/Recovery time	S9 activation	Mean mitotic index	Cells scored	Aberrant cells [%]				Aberrations per cell
					Numerical			structural	Mean	SD
					Poly	Endo	Total
Ethanol	4/16	-	9.2	200	1.5	0.0	1.5	1.5	0.015	0.122
Test item 50	4/16	-	9.3	200	1.0	0.0	1.0	3.5	0.035	0.184
Test item 100	4/16	-	9.0	200	2.5	0.0	2.5	4.0	0.040	0.196
Test item 200	4/16	-	7.6	200	3.5	0.0	3.5	3.5	0.040	0.221
MMC, 0.2	4/16	-	8.9	200	1.5	0.0	1.5	18.5**	0.225	0.506
Ethanol	4/16	+	13.0	200	2.0	0.0	2.0	4.0	0.040	0.196
Test item 50	4/16	+	12.7	200	2.0	0.0	2.0	4.5	0.045	0.208
Test item 100	4/16	+	11.3	200	2.0	0.0	2.0	6.0	0.060	0.238
Test item 200	4/16	+	10.0	200	2.0	0.0	2.0	3.5	0.035	0.184
CP, 10	4/16	+	6.5	200	2.0	0.0	2.0	31.0**	0.515	0.902
Ethanol	20/0	-	13.4	200	1.5	0.0	1.5	2.0	0.020	0.140
Test item 50	20/0	-	13.0	200	2.0	0.0	2.0	4.0	0.040	0.196
Test item 100	20/0	-	11.0	200	1.5	0.0	1.5	3.5	0.035	0.184
Test item 200	20/0	-	11.3	200	1.5	0.0	1.5	4.0	0.040	0.196
MMC, 0.1	20/0	-	10.2	200	1.0	0.0	1.0	18.5**	0.205	0.452

 

Mitotic index= cells in mitosis per 500 cells counted, expressed as a percentage.

% Aberrant Cells:significantly increased above the solvent control using Fisher's Exact test with Bonferroni adjustment, * = p</=0.05; ** = p</=0.01.

Structural aberrations per cell:include unanalyzable cells and cells with one or more aberrations, excluding gaps.

Applicant's summary and conclusion

Conclusions:

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.