Ethanol, 2,2'-oxybis-, 1,1'-diformate

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal 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:

2,2'-oxybis-ethanol diformate

EC Number:

601-722-4

Cas Number:

120570-77-6

Molecular formula:

C6H10O5

IUPAC Name:

2,2'-oxybis-ethanol diformate

Details on test material:

- Name of test material (as cited in study report): 2,2-Oxybis-ethanol diformate (Diethylene Glycol Diformate)
- Analytical purity: no data

For OECD 471, OECD 473, OECD 490, OECD 414 and OECD 408
Name of test material (as cited in the tstudy report): Diethylene glycol diformate
CAS No. 120570-77-6
Chemical Name (IUPAC) : 2,2’-oxybis-ethanol diformate:
Physical Appearance : Clear pale-yellow liquid, free from suspended matter
Purity (as per CoA) : 96.1%
Batch No. : 26088
Manufactured Date : 07 July 2019
Retest Date : 06 July 2021
Recommended Storage Condition: Ambient(+18 to +36ºC)
pH: 2.8 at 30% water solution
density: 1.19 at 20°C

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Blood was collected f rom male donors of age 24 and 30 years old for preliminary citotoxicity test and chromosomal aberation tests respectively, who were no smokers, not undergoing any drug treatment and not suspected of exposure to high levels of radation or hazardous chemicals or any viral infection

Cytokinesis block (if used):

colchicine at 0.2 microg/ml of final concentration in the culture media was used as the spindle inhibitor

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix:
The S9 homogeneate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 , 5 days prior to sacrifice. The S9 homogeneate was prepared in batches and stored in a deep freezer mantained at -68 to -86°. The S9 homogeneate was thawed immediately before use and mixed with the co-factor solution, containing 25 mg/ml NADP, 180 mg7ml Glucose-6-phosphate, 150 mM KCl, and S9 fraction (ratio 1:1:1:2 v/v)

Test concentrations with justification for top dose:

500, 1000 and 2000 mcrog/ml with and without metabolic activation for 3h exposure
500, 1000 and 2000 microg/ml without metabolic activation for 22 hours exposure
the test concentrations were based on the results of a preliminary citotoxicity test with the same conditions (with and without metabolic activation for 3h exposure, without metabolic activation for 22 hours exposure) at the dose 62.5, 125, 250, 500, 1000 and 2000 microg/ml that resulted at the highest dose level in a reduction in mitotic index by 45+/-5% of the solvent control.

Vehicle / solvent:

sterile water

Controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : three

METHOD OF TREATMENT/ EXPOSURE:
the target cells were exposed to the controls and three concetrations of the test itme as follows:
Experiment 1 and 3: the target cells in duplicate were exposed to the vechilce control positive control and test concentrations for 3h in the presence and for 22h in the absence of metabolic activation, respectively
Experiment 2: the target cells in duppkicate were exposed to the vehicle control and the test concentrations for 3h in the absence of metabolic activation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h with and without, 22h without metabolic activation
- Harvest time after the end of treatment (sampling/recovery times): each culture from the controls and treatmenet groups was harvested at approx 22h after beginning of treatment and processed separately for preparation of chromosomes.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine at 0.2 microg/ml of final concentration in the culture media was added approximately 2h before harvest

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): cells were pelleted and re-suspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of supsension were transferred on to clean glass slides, flame dried and dried on a slide warmer mainted at apprx. 40°C. Five slides per replicate were made.
The sldes were stained with freshly prepared 5% Giemsa stain in distilled water, rinsed with tap water , ari dreid, immersed in xylene and mounted with DPX.

- number of cells scored: 150 metaphases were scored from each replicate for chromosome aberrations.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The number of chromosomes for each spread was counted , and those containing 44 to 48 chromosome were evaluated for aberration. The chromosome number was recorded for all cells analysed and the microscope coordinates were recorded for the aberrant cells. Aberrations were recorded as chromatid/chromosome gaps or breaks and exchanging figures.Since gaps are not considered as true aberrations, the results are presented as metaphases with aberration includinf gaps and excluding gaps.
- Determination of polyploidy:
- Determination of endoreplication:


METHODS FOR MEASUREMENT OF CYTOTOXICITY
-concurrent cytotoxicity was assesed for all treated and control cultures based on mitotic index


METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

whne all the validaity criteria are fulfilled:
-A test chemicl is considered to be clearly positive if in any of the experimental conditions examined: 1) at least one of the test concentrations exhibits a statistically significant increase compared with concurrent vehicle control 2)the increase is dose-dependent when evaluated with an appropriate trend test 3)any of the results are outside the distribution of the historical vehicle control data
-A test chemicl is considered to be clearly negative if in all of the experimental conditions examined: 1) None of the test concentrations exhibits a statistically significant increase compared to the concurrent vehcile control 2)there is not concentration related increase when evaluated with an appropriate trend test 3)all results are within the distribution of the historical vehicle control data
-the results will be considered equivocal if they do no meet the criteria specified for a positive or a negative response
-An increase in the number of polyploidy cells may indicate that the test item has the potential to inhibit mitotic process and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhiit cell cycle progression.

Statistics:

The statistical analysis was performed via SYSTAT statistical package version 12.0.Data were analysed for proportions of aberrant metaphases in each sample, excluding gaps as aberrations. Pooled data from each test concentrations and the positive control were compared with the vehicle control uing Fischer exact test. All analyses and comparison were evaluated at 5% (p<0.05) level

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral blood

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:

Experiment 1: at the highest tested dose (2000 microg/ml) the mitothic inhibition was 41% compared to the vehicle control
The incidence of aberrations of the vehicle control was within the range of the in-houe historical control data
There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control cyclophosphamide monohydrate caused a statistically significant increase in the aberrant metaphases excluding gaps.

Experiment 2: at the highest tested dose (2000 microg/ml) the mitothic inhibition was 40% compared to the vehicle control
The incidence of aberrations of the vehicle control was within the range of the in-houe historical control data
There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen.

Experiment 3: at the highest tested dose (2000 microg/ml) the mitothic inhibition was 45% compared to the vehicle control
The incidence of aberrations of the vehicle control was within the range of the in-houe historical control data
There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control ethylmethanesulphonate caused a statistically significant increase in the aberrant metaphases excluding gaps.

Any other information on results incl. tables

refer to the attached full study report for tables and details

Applicant's summary and conclusion

Conclusions:

The substance was tested for in vitro citogenicity following OECD 473. Under the experimental conditions no evidence of induction of chromosome aberrations excluding gaps was obtained in any of the experiments at any test concentrations either in the presence or in the absence of metabolic activation.Similarly there were no incidences of polyploidy and endoreduplications.
It i concluded that the test item was not clastogenic in cultured human peripheral blodd lymphocytes up to the respective highest concentraion of 2000 microg/ml.

Executive summary:

The clastogenic potential of the test item Diethylene Glicole Diformate to induce chromosome aberration in mammalian cells was evaluted using cultured human peripheral blood lymphocytes.

Human lymphocytes in the blood cultre stimulated to divide by addition of PHA 48h prior to treatment, were exposed to the test item in the presence and absence of an exogenous metabolic activation sysmte (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted in a preliminary cytotoxicity test and a chromosomal aberration assay. The latter consisted of three indipendent experiments: Experiment 1 and 2 in the presence and absence of metabolic activation sysem and 3h exposure, and Experiment 3 in the absence of metabolic activation with a 22h expoure.

The test item was soluble in sterile water at 200 mg/ml and was found to be stable in the Sterile water for 3h at room temperature and fortification levels of 300 microg/ml and 200000 microg/ml.

In a preliminary cytotoxicity test for the selection for the test concentrations for the chromosomal aberration assay, DEGDF exhibited the required level of cytotoxicity (reduction in the mitothic index by 45 +/- 5% of the concurrent vehicle control) at the highest tested concentration both in the presence and absence of metabolic activation with 3h expoure and also in the absence of metabolic activation with 22h exposure at and up to 2000 microg/ml. Based on these observation, in the chromosomal aberration assay, a top concentration of 2000 microg/ml was tested in the 3h exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation with 22h exposure.

In the definitive chromosomal aberration assay, bllod cultures were exposed to the test item in duplicate at the concentration of 500, 1000 and 2000 microg/ml in Experiment 1 and 2 (3h exposure with and without metbaolic activation) and in Experiment 3 (22h exposure without metabolic activation).

In a similar way concurrent vehicle control and the positive controls (cyclophophamide monohydrate in the presence of metabolic activation and ehtylmethane sulfonate in the absecne of metabolic activation) were tested in duplicate cultures. In each case the cells in the c-metaphase were harvsted at approximately 22h after the start of treatment from the vehicle control, test item concentrationd and positive controls.

At the highest concentration tested, the reduction in mitothic index was 41, 40 and 45% in Experiments 1, 2 and 3 respectively, compared to the vehicle control.

A total of 300 metaphases from duplicate cultures from the controls and threee treatment levels were evaluated for chomosomal aberrations. The data from the treatment groups and the positive controls were statisically compared with the vehicle control. There were not statistically significant increase in the incidence of structually aberrant metaphases , either in the presence or in the absence of metabolic activation at any tested concentrations. Under identical conditions the respective positive controls produced statistically significant (p < 0.05) increases in aberrant metaphases confirming the sensitivity of the test item and the activity of the S9 mix.

The results of the concentration analyses were of the dose formulation samples were within the acceptable specification range and confirmed that the targeted top concentration level and the results support the validity of the study conclusion.

Under the experimental conditions DEGDf was not clastogenic in hman perypheral blood lymphocytes up to 2000 microg/ml.