Fatty acids, C18-36, esters with ethylene glycol

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:

09 June 2017 to 12 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011

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:

Fatty acids, C18-36, esters with ethylene glycol

EC Number:

305-673-7

EC Name:

Fatty acids, C18-36, esters with ethylene glycol

Cas Number:

94944-95-3

Molecular formula:

C40H78O4/C70H138O4

IUPAC Name:

Fatty acids, C18-36, esters with ethylene glycol

Test material form:

solid

Method

Target gene:

NA

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human peripheral lymphocytes
- Suitability of cells: standard cells (checked for exposure to high levels of radiation or hazardous chemicals and not knowingly recently suffered from a viral infection)
- Cell cycle length, doubling time or proliferation index: AGT 16 hours
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: male, aged 31 years; Main Experiment: female, aged 30 years
- Whether whole blood or separated lymphocytes were used if applicable: fresh heparinized whole blood
- Methods for maintenance in cell culture: grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air
- Modal number of chromosomes: 44-48
- Normal (negative control) cell cycle time: 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration: MEM at approximately 37 ºC with 5 % CO2
- Properly maintained: yes

Cytokinesis block (if used):

demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time.

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat livers induced with phenobarbital/beta-naphthoflavone

Test concentrations with justification for top dose:

Exposure Group Final concentration of

4(20)-hour without S9 0*, 1.25, 2.5, 5, 8*, 10*, 20*, 40, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 5, 8, 10, 15*, 20*, 40*, 80, CP 1*
24-hour without S9 0*, 1.25, 2.5, 5, 8*, 10*, 20*, 40, MMC 0.1*


* Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)(0.25% in 25 µl aliquots; maximum achieved dosing concentration 625 µg/mL)
- Justification for choice of solvent/vehicle: insoluble in dimethyl sulphoxide at 125, 250 and 500 mg/mL and acetone at 250 and 500 mg/mL. However, the substance was soluble in THF at 500 mg/mL

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- pre-incubation: 48 hours at approximately 37 ºC, 5% CO2 in humidified air
- Exposure duration: 4 hour with and without metabolic activation, 24 hours without metaboliv activation at approximately 37 ºC, 5% CO2 in humidified air
- Expression time (cells in growth medium): 4 hour tests: 20 hours at approximately 37 ºC, 5% CO2 in humidified air

SPINDLE INHIBITOR: Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2/concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 2000/concentration for mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE : 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells
- Determination of endoreplication: recorded separately and included in the polyplod cell total

Rationale for test conditions:

standard according to the guidance

Evaluation criteria:

•The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
•All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
•The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
•The required number of cells and concentrations were analyzed

Statistics:

Fisher's Exact test (p<0.05)

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: see vehicle selection
- Precipitation: yes at 20 ug/mL in tests without metabolic activation and at 40 ug/mL in test with metabolic activation

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available (all findings were within historical control values)
- Negative (solvent/vehicle) historical control data: available (all findings were within historical control values)

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity observed

Any other information on results incl. tables

details see attached document

Applicant's summary and conclusion

Conclusions:

The substance is considered not clastogenic under the conditions of the test

Executive summary:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were as follows:

Group	Final concentration of substance evaluated
4(20)-hour without S9	0, 8, 10, 20
4(20)-hour with S9 (2%)	0. 15, 20, 40
24-hour without S9	0, 8, 10, 20

All vehicle (Tetrahydrofuran) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. 

The test item,was considered to be non-clastogenic to human lymphocytes in vitro.