[No public or meaningful name is available]

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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:

Methyl N-octadecylterephthalamate

EC Number:

230-836-3

EC Name:

Methyl N-octadecylterephthalamate

Cas Number:

7333-86-0

Molecular formula:

C27H45NO3

IUPAC Name:

methyl 4-(octadecylcarbamoyl)benzoate

Constituent 2

Reference substance name:

Dimethyl terephthalate

EC Number:

204-411-8

EC Name:

Dimethyl terephthalate

Cas Number:

120-61-6

Molecular formula:

C10H10O4

IUPAC Name:

1,4-dimethyl benzene-1,4-dicarboxylate

Test material form:

solid: flakes

Details on test material:

UVCB, C(16-18) amides of methyl terephthalamate. EC # 934-047-1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus Holdings LLC, Batch 48654
- Expiration date of the lot/batch: 17 May 2020
- Purity test date: N/A

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under test conditions: Assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: 20 minute sonication at 37 degrees C required to dissolve, stable for at least 4 hours
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 1 mg/ml
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : N/A

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS:

Method

Target gene:

N/A

Species / strain

Species / strain / cell type:

lymphocytes:

Remarks:

Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 mix containing MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4)

Vehicle / solvent:

cell culture medium

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 500 µL heparinized whole blood in 4.5 mL complete culture medium. Culture medium: RPMI 1640 supplemented with 15% FBS, 100 U/100 µg/ml penicillin/streptomycin solution, 0.24 g/ml PHA-L.

DURATION : See table

ANALYSIS OF METAPHASE CELLS: Evaluation of the cultures is performed [according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik"] using microscopes with 100x oil immersion objectives.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN: BrdU

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): At least 300

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cultures are harvested by centrifugation 24 h after beginning of treatment. The supernatant is discarded and the cells are resuspended in approximately 5 mL hypotonic solution (0.4 % KCl). The cell suspension is incubated at room temperature for 20 min. After removal of the hypotonic solution by centrifugation the cells are fixed with 3+1 methanol + glacial acetic acid. The fixation procedure is repeated twice. Slides are prepared by dropping the cell suspension onto a clean microscopic slide. The cells are stained with giemsa and according to the Fluorescent plus Giemsa technique, respectively. The slides are coverslipped using 2-3 drops of Eukitt(R). Afterwards they are air dried.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: The number of polyploid cells is scored

OTHER EXAMINATIONS:
- Determination of polyploidy: Near tetraploid karyotype
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER:

Evaluation criteria:

All structural chromosomal aberrations including breaks, fragments, deletions, exchanges and chromosomal disintegration are recorded. Gaps are recorded as well but not included in the calculation of the aberration rates. The definition of a gap is as follows: an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated either longitudinally or laterally.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured human peripheral blood lymphocyte cells in this test system.

Statistics:

Not reported

Results and discussion

Test results

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: precipitation at highest dose(s)

Applicant's summary and conclusion

Conclusions:

The test substance was evaluated for potential to cause chromosome aberrations using an in vitro Chromosome Aberration (CA) Test according to OECD 473 in human lymphocytes. The test substance was determined to be non-cytogenic according to the results of this test.