Phosphoric acid C12-alkyl and C18-alkyl esters, potassium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: other: clastogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-14 to 2012-02-23

Reliability:

1 (reliable without restriction)

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:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

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:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, dodecyl ester, potassium salt

EC Number:

254-414-3

EC Name:

Phosphoric acid, dodecyl ester, potassium salt

Cas Number:

39322-78-6

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of potassium didodecylphosphate and dipotassium dodecylphosphate and water

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 15.6, 250, 500 and 1000 µg/mL
with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:
without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL
with metabolic activation: 900, 1600 and 1800 µg/mL

Vehicle / solvent:

-Vehicle (s)/solvent(s) used: cell culture medium (MEM)
-Justification for choice of solvent/vehicle: The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: 400 and 900 µg/mL

Controls 2

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation Migrated to IUCLID6: 0.83 µg/mL

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 300 cells
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results of chromosome analysis
without metabolic activation
		Cytotoxicity	Chromatid aberrations				Isochromatid aberrations				rel. Mitotic index (%)	rel. Cell density (%)	Poly-ploidy	mean % aberrant cells
	Scored cells		gaps	breaks	inter-changes	other	gaps	breaks	inter-changes	other				incl. Gaps	excl. Gaps
Experiment I
negative control	200	-	5	4	0	0	0	0	1	1	100	100	2	4.5	2.5
15.6 µg/mL	200	no	5	0	0	0	0	0	0	0	103	96	3	2.5	0.0
250 µg/mL	200	no	6	2	0	1	0	0	0	0	81	78	3	3.5	1.5
500 µg/mL	200	yes	4	3	2	0	0	0	2	0	53	55	1	5.0	3.0
1000 µg/mL	200	yes	4	1	0	0	1	0	0	0	51	44	0	3.0	0.5
EMS (900 µg/mL)	200	-	7	10	8	2	0	1	2	0	85	94	3	12.5	9.5
Experiment II
negative control	200	-	1	4	0	0	0	0	0	0	100	100	2	2.5	2.0
15.6 µg/mL	200	no	6	3	0	1	0	0	0	0	76	102	3	4.5	2.0
31.3 µg/mL	300	no	5	7	1	1	0	0	0	0	74	94	1	4.7	3.0
62.5 µg/mL	200	yes	2	2	0	0	0	0	1	0	51	89	1	2.5	1.5
125.0 µg/mL	200	yes	1	2	0	0	0	0	1	0	30	57	1	2.0	1.5
EMS (400 µg/mL)	200	-	7	17	3	3	3	0	0	0	72	103	0	13.0	10.5
Results of chromosome analysis
with metabolic activation
		Cytotoxicity	Chromatid aberrations				Isochromatid aberrations				rel. Mitotic index (%)	rel. Cell density (%)	Poly-ploidy	mean % aberrant cells
	Scored cells		gaps	breaks	inter-changes	other	gaps	breaks	inter-changes	other				incl. Gaps	excl. Gaps
Experiment I
negative control	200	-	6	2	0	0	0	0	0	0	100	100	0	4.0	1.0
15.6 µg/mL	300	no	4	3	3	1	0	0	2	0	96	102	1	4.0	3.0
500 µg/mL	200	no	2	0	2	0	0	0	0	0	90	86	0	2.0	1.0
1000 µg/mL	200	yes	3	0	0	0	0	0	0	0	62	85	1	1.5	0.0
1500 µg/mL	200	yes	2	1	0	0	1	0	0	0	54	63	1	2.0	0.5
CPA (0.83 µg/mL)	200	-	3	12	11	1	0	1	1	0	104	85	3	12.0	10.5
Experiment II
negative control	200	-	4	5	0	1	1	0	1	0	100	100	2	5.5	3.5
900 µg/mL	200	no	1	3	0	0	0	0	0	0	71	88	0	2.0	1.5
1600 µg/mL	200	yes	3	3	0	0	0	0	0	0	54	75	0	2.5	1.5
1800 µg/mL	200	yes	0	2	0	0	0	0	0	1	45	56	0	1.5	1.5
CPA (0.83 µg/mL)	200	-	3	9	3	3	1	1	2	1	96	100	0	10.0	9.0

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item (CAS39322-78-6) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of Afilan V5756 to induce structural chromosome aberrations in Chinese hamster V79 cells, anin vitrochromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.

Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations, except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 200 cells for the 1st culture and 100 cells for the 2nd culture.

The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 15.6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

with metabolic activation: 900, 1600 and 1800 µg/mL

Precipitation of the test item was observed with and without metabolic activation in both experiments.

Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

The positive controls induced the appropriate responses.

There was no evidence of Afilan V5756 induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data.