1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 24 February to 21 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

N° 2011/40

Type of assay:

other: in vitro mammalian aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts

EC Number:

939-455-3

Molecular formula:

Molecular formula, SMILES notation, InChl and Structural formula cannot be given as the substance is a UVCB.

IUPAC Name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: Cultures of human lymphocytes were prepared from whole blood samples obtained from healthy, non-smoking donors and collected into heparinized sterile tubes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.

Test concentrations with justification for top dose:

1st experiment without S9 mix (3h treatment): 0; 39.06; 78.13; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL
2nd experiment without S9 mix (20h treatment): 0; 9.38; 18.18; 37.5; 75; 150; 300; 600 µg/mL
2nd experiment without S9 mix (44h treatment): 0; 9.38; 18.18; 37.5; 75; 150; 300; 600 µg/mL
1st experiment with S9 mix (3h treatment): 0; 39.06; 78.13; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL
2nd experiment with S9 mix (3h treatment): 0; 9.4; 18.8; 37.5; 75; 150; 300 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injections, batch No. 1F1731 (CDM Lavoisier, France)
- Justification for choice of solvent/vehicle: based on solubility data for test substance

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

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: not applicable
- Exposure duration: without Sç mix: 3; 20 or 44h / with S9 mix: 3h
- Expression time (cells in growth medium): 20 or 44h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 or 44h

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid (10µg/mL) added 3h before the harvest time
STAIN (for cytogenetic assays): After harvest, the cells were collected by centrifugation and submitted to a hypotonic treatment (KCl 0.075 M). The cells were then fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.

NUMBER OF REPLICATIONS: 2 independent experiments, 2 cultures in parallel (same donor) per concentration

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level (on metaphases which contained 44 to 46 chromosomes). Whenever possible, 100 metaphases were scored for each culture. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (The number of cells in mitosis is scored on a total of 1000 cells per culture)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER: according to the purity of the test substance (36.2%), a correction factor of 2.76 was applied to prepare the substance to be tested in order to assess thegenotoxicity of the substance at 100% (in accordance with the REACh requirements).
For each experiment, both cultures were prepared from the blood of one distinct donor.

Evaluation criteria:

This study was considered valid since the following criteria were met:
- the frequency of cells with structural chromosome aberration in the vehicle controls was consistent with (but not necessary within) the historical data. In any case this frequency was ≤ 5%,
- the frequency of cells with structural chromosome aberration in the positive controls was significantly higher than that of the vehicle controls (p ≤ 0.05) and consistent with (but not necessary within) the historical data.

A test item is considered positive for inducing chromosomal aberrations if a reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration is observed at one or more dose levels and at one or two harvest times.
A test item is considered negative for inducing chromosomal aberrations if no significant increase is observed in the number of cells with chromosomal aberrations for any of the dose levels and at any harvest times.

Statistics:

For each experiment and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the Che 2 test unless treated culture data were lower than or equal to the vehicle control data. p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: At 5000 µg/mL, the pH of the culture medium was approximately 7.1 (7.4 for the vehicle control)
- Effects of osmolality: At 5000 µg/mL, the osmolality was equal to 319 mOsm/kg H2O (289 for the vehicle control).
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitate was observed at the end of the treatment periods at any dose levels.

RANGE-FINDING/SCREENING STUDIES: no range-finding study was performed

COMPARISON WITH HISTORICAL CONTROL DATA:The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9 mix:
Following the 3-hour treatment, a slight to severe toxicity was observed at all dose-levels, as shown by a 35-100% decrease in the mitotic index.
Following the 20-hour treatment, a slight to severe toxicity was noted at dose-levels ≥ 150 µg/mL, as shown by a 39-100% decrease in the mitotic index.
Following the 44-hour treatment, a moderate to severe toxicity was noted at dose-levels ≥ 150 µg/mL, as shown by a 43-100% decrease in the mitotic index.
- With S9 mix:
At the 20-hour harvest time in the first experiment, a moderate to severe toxicity was observed at dose-levels ≥ 78.13 µg/mL, as shown by a 42-100% decrease in the mitotic index.
At the 20-hour harvest time in the second experiment, a slight to severe toxicity was observed at dose-levels ≥ 9.4 µg/mL, as shown by a 30-99% decrease in the mitotic index.
At the 44-hour harvest time, a slight to severe toxicity was observed at dose-levels ≥ 18.8 µg/mL, as shown by a 32-92% decrease in the mitotic index.

Any other information on results incl. tables

Results of chromosome analysis for the experiment 1 without S9 mix (3h treatment)

Dose Level (µg/mL)		Control		Low dose (78.3)		Mid dose (156.3)		High dose (312.5)		Positive control (3µg/mL)
Cytotoxicity		no		yes		yes		yes		yes
Culture		C1	C2	C1	C2	C1	C2	C1	C2	C1	C2
Chromatid aberrations	breaks	0	0	0	0	0	0	1	4	20	18
	interchanges	0	0	0	0	0	0	0	0	6	6
Isochromatid aberrations	breaks	0	0	0	0	0	0	0	0	4	3
	interchanges	0	0	0	0	0	0	0	0	0	0
Multiple aberrations		0	0	0	0	0	0	0	0	4	3
Pulverization		0	0	0	0	0	0	0	0	0	0
Total number of structural aberration (- gap)		0		0		0		5		64
Frequency of Cells with structural chromosome aberrations (- gap)		0%		0%		0%		2%		39%
Mitotic index		100%		62%		56%		44%		36%
Numerical aberrations		1	0	0	0	1	0	1	0	0	0

Results of chromosome analysis for the experiment 1 with S9 mix (3h treatment)

Dose Level (µg/mL)		Control		Low dose (39.06)		Mid dose (78.3)		High dose (156.3)		Positive control (12.5µg/mL)
Cytotoxicity		no		yes		yes		yes		yes
Culture		C1	C2	C1	C2	C1	C2	C1	C2	C1	C2
Chromatid aberrations	breaks	0	0	0	0	0	0	0	1	12	21
	interchanges	0	0	0	0	0	0	0	0	2	3
Isochromatid aberrations	breaks	0	0	0	0	0	0	0	0	8	3
	interchanges	0	0	0	0	0	0	0	0	0	0
Multiple aberrations		0	0	0	0	0	0	0	0	0	0
Pulverization		0	0	0	0	0	0	0	0	0	0
Total number of structural aberration (- gap)		0		0		0		1		49
Frequency of Cells with structural chromosome aberrations (- gap)		0%		0%		0%		0.5%		32%
Mitotic index		100%		81%		58%		44%		62%
Numerical aberrations		0	0	0	0	1	0	2	0	0	0

Results of chromosome analysis for the experiment 2 without S9 mix (20h treatment)

Dose Level (µg/mL)		Control		Low dose (78.3)		Mid dose (150)		High dose (300)		Positive control (0.3µg/mL)
Cytotoxicity		no		yes		yes		yes		no
Culture		C1	C2	C1	C2	C1	C2	C1	C2	C1	C2
Chromatid aberrations	breaks	0	0	1	0	1	2	3	0	5	8
	interchanges	0	0	0	0	0	0	0	1	2	0
Isochromatid aberrations	breaks	0	0	0	0	0	0	0	1	1	0
	interchanges	0	0	0	0	0	0	1	0	0	0
Multiple aberrations		0	0	0	0	0	0	0	0	0	0
Pulverization		0	0	0	0	0	0	0	0	0	0
Total number of structural aberration (- gap)		0		1		3		6		16
Frequency of Cells with structural chromosome aberrations (- gap)		0%		0.5%		1.5%		2.5%		14%
Mitotic index		100%		89%		61%		39%		110%
Numerical aberrations		1	0	3	7	1	3	2	3	1	0

Results of chromosome analysis for the experiment 2 without S9 mix (44h treatment)

Dose Level (µg/mL)		Control		300
Cytotoxicity		no		yes
Culture		C1	C2	C1	C2
Chromatid aberrations	breaks	2	0	2	0
	interchanges	0	0	0	0
Isochromatid aberrations	breaks	1	1	1	2
	interchanges	0	0	0	0
Multiple aberrations		0	0	0	0
Pulverization		0	0	0	0
Total number of structural aberration (- gap)		4		5
Frequency of Cells with structural chromosome aberrations (- gap)		2%		2%
Mitotic index		100%		43%
Numerical aberrations		0	0	7	10

Results of chromosome analysis for the experiment 2 with S9 mix (3h treatment)

Dose Level (µg/mL)		Control		Low dose (9.4)		Mid dose (18.8)		High dose (37.5)		Positive control (12.5µg/mL)
Cytotoxicity		no		yes		yes		yes		yes
Culture		C1	C2	C1	C2	C1	C2	C1	C2	C1	C2
Chromatid aberrations	breaks	1	1	0	1	4	0	1	0	15	14
	interchanges	0	0	0	0	0	0	0	0	6	5
Isochromatid aberrations	breaks	1	0	0	1	0	0	0	0	1	4
	interchanges	0	0	0	0	0	0	0	0	0	0
Multiple aberrations		0	0	0	0	0	0	0	0	0	0
Pulverization		0	0	0	0	0	0	0	0	0	0
Total number of structural aberration (- gap)		3		2		4		1		45
Frequency of Cells with structural chromosome aberrations (- gap)		1.5%		1%		1.5%		0.5%		32%
Mitotic index		100%		65%		70%		50%		38%
Numerical aberrations		0	0	2	2	1	1	7	11	0	0

Applicant's summary and conclusion

Conclusions:

The study was negative for structural chromosome aberrations. However numerical chromosome aberrations were observed with and without metabolic activation. Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not induce structural chromosome aberrations in cultured human lymphocytes, exposed for 3 hours to up to 156.3 µg/mL in the presence of a rat metabolizing system, or exposed for 3 hours to up to 312.5 µg/mL and for up to 44 hours to up to 300 µg/mL in the absence of metabolizing system.

Executive summary:

In an in vitro chromosome aberration study, performed according to OECD 473 and in compliance with GLP, Cocamidopropyl hydroxysultaine (as an aqueous solution of purity 36.2%) diluted in water was tested in cultured human lymphocytes in the presence and the absence of exogenous mammalian metabolic activation (S9 mix). The concentrations used for treatments were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment, both with and without S9 mix; 9.38, 18.8, 37.5, 75, 150, 300 and 600 µg/mL for the second experiment without S9 mix, and 9.4, 18.8, 37.5, 75, 150 and 300 µg/mL for the second experiment with S9 mix. The amount used for the test was calculated as concentration of Cocamidopropylhydroxysultaine and therefore a correction factor of 2.76 was applied. Without metabolic activation, cells were exposed to the test substance for 3 (exp 1), 20 or 44h (exp 2) whereas with metabolic activation the treatment period was of 3 hours in both experiments. In Experiment 1 without S9 mix and in both experiments with S9 mix, cells were rinsed after the 3hrs of treatment with the test substance and placed in fresh medium culture until the harvest time. Cells were harvested 20 or 44h after the beginning of the experiment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. Three hours before harvest, each culture was treated with a Colcemid solution to block cells at the metaphase-stage of mitosis. Analysis for clastogenicity and aneuploidy was undertaken on 200 (100 per culture; 2 parallel cultures) metaphases/concentration (on metaphases which contained 44 to 46 chromosomes). Cytotoxicity of the test substance was assessed by the mitotic index: the number of cells in mitosis was scored on a total of 1000 cells per culture. Positive controls such as mitomycin C and Cyclophosphamid were used to check the sensitivity of the test system. They gave appropriate response, so that the test was considered as valid. The test substance induced cytotoxicity. Indeed, a decrease of mitotic index was observed in both experiments with and without metabolic activation. The highest tested dose level for metaphase analysis induced around 50% cytotoxicity. Cocamidopropyl hydroxysultaine did not induce structural chromosome aberrations in cultured human lymphocytes with and without metabolic activation at any treatment time. In the second experiment only, increases in the numerical aberrations were noted when compared to the vehicle control cultures. These numerical aberrations exclusively consisted of polyploidy. However, the relevance of such findings was limited as they were observed without any clear evidence of a dose-response relationship or consistency between cell cultures. Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not induce structural chromosome aberrations in cultured human lymphocytes, exposed for 3 hours to up to 156.3 µg/mL in the presence of a rat metabolizing system, or exposed for 3 hours to up to 312.5 µg/mL and for up to 44 hours to up to 300 µg/mL in the absence of the metabolizing system.