Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-02-08 to 2016-04-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

EC Number:

915-372-8

Molecular formula:

not applicable (multi-const.substance)

IUPAC Name:

Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Lot. No.: 10H016
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Modal number of chromosomes: 2n = 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated fetal bovine serum (final concentration 10 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Concentrations were chosen based on a preliminary cytotoxicty test:
Experiment A
150, 125, 100, 50 µg/mL
Experiment B
75, 50, 25, 12.5, 1.5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^5 cells/dish

DURATION
- Exposure duration: 3 h with metabolic activation; 20 h without metabolic acitvation
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): 1.5 cell cycles (20 h, without S9 mix only) and at approximately 2 normal cell cycles (28 h, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

SPINDLE INHIBITOR: colchicine (0.2 μg/mL) 2.5-3 hours prior to harvest.

STAIN: 5 % Giemsa

NUMBER OF REPLICATIONS: 2 plates/concesntration

NUMBER OF CELLS EVALUATED: 300

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)
- Any supplementary information relevant to cytotoxicity:

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

Evaluation criteria:

– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations were listed with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) were recorded.
– Individual culture data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Statistics:

For statistical analysis, Fisher exact and CHI2 tests were utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

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

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: no differences to negative control
- Effects of osmolality: no differences to negative control
- Precipitation: There was no precipitation in the medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see table 1. - 5. in "any other information on results"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Any other information on results incl. tables

Table 1. 3 h/20 h treatment/sampling time without S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Ethyl methanesulfonate)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	4.59	2.16	39.84	32.16
SD	1.84	1.37	5.01	4.02
Lower confidence interval	0.00	0.00	23.89	19.38
Upper confidence interval	10.44	6.52	55.80	44.94
n	4	4	4	4

 

Table 2. 3 h/20 h treatment/sampling time with S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Cyclophosphamide)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	5.44	2.34	50.34	44.53
SD	1.73	0.62	2.53	4.35
Lower confidence interval	0.00	0.38	42.31	30.71
Upper confidence interval	10.95	4.31	58.38	58.36
n	4	4	4	4

 

Table 3. 20 h/20 h treatment/sampling time without S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Ethyl methanesulfonate)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	4.94	2.34	45.28	39.75
SD	1.06	0.79	4.11	2.55
Lower confidence interval	1.55	0.00	32.21	31.64
Upper confidence interval	8.32	4.87	58.35	47.86
n	4	4	4	4

 

 

Table 4. 20 h/28 h treatment/sampling time without S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Ethyl methanesulphonate)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	5.06	2.16	45.94	40.69
SD	0.87	0.36	4.61	12.43
Lower confidence interval	2.31	1.01	31.27	28.26
Upper confidence interval	7.82	3.03	60.61	53.11
n	4	4	4	4

 

Table 5. 3 h/28 h treatment/sampling time with S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Cyclophosphamide)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	4.97	2.16	47.91	40.59
SD	0.36	0.94	2.81	3.10
Lower confidence interval	3.82	0.00	38.97	30.72
Upper confidence interval	6.12	5.14	56.84.	50.47
n	4	4	4	4

 

Applicant's summary and conclusion

Conclusions:

The test item tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.
Therefore, the test item is considered as not clastogenic in this system.

Executive summary:

The test item, was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 50, 100, 125 and 1501 μg/mL

with S9 mix: 100, 150, 175 and 200 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 12.5, 25, 50 and 751 μg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 12.5, 25, 50 and 75 1 μg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 100, 150, 175 and 200 μg/mL

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.