Reaction products of ethylene glycol, urea and paraformaldehyde (EUF)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 13, 2004 to January 20, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test performed according to generall accepted protocols: Tice et al. (2000) Single Cell Gel/Comet Assay: Guidelines for In Vitro und In Vivo Genetic Toxicology Testing. Environ. Mol. Mutagen. 35(3):206-221

GLP compliance:

yes (incl. QA statement)

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

reaction products of ethylene glycol, urea and paraformaldehyde

EC Number:

700-934-5

Molecular formula:

No exact molecular formula can be given for a complex reaction mixture (UVCB substance).

IUPAC Name:

reaction products of ethylene glycol, urea and paraformaldehyde

Details on test material:

- Name of test material (as cited in study report): EUF
- Substance type: Formaldehyde releaser
- Physical state: Clear colourless fluid, density (20°C) 1.256 g/mL
- Analytical purity: Reaction product of urea, ethylene glycol and formaldehyde. Therefore purity is not exactly known. Releasable formaldehyde: 46.1%
- Composition of test material, percentage of components: Reaction product of urea, ethylene glycol and formaldehyde. Main component: (Ethylenedioxy)-dimethanol (CAS 3586-55-8)
- Lot/batch No.: LJ 526/37
- Expiration date of the lot/batch: December 2006
- Storage condition of test material: at room temperature in a well closed container

Method

Target gene:

DNA in the mouse lymphoma cell line L5178Y/TK+/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-liver homogenate

Test concentrations with justification for top dose:

With EMS: 25, 62.5, 125, 250, and 500 µM
Without EMS: 2.5, 25, and 250 nM; 2.5, 25, and 250 µM
Concerning EUF, concentrations in the main experiments represent a hypothetical FA content of the aqueous solutions, based on FA liberation of about 19.5 % (w/w)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Medium containing 0.02% methanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Formaldehyde (FA):, reference and positive control item for cross-links; 2-Aminoanthracene (AA): positive control for S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Exponentially growing suspension cultures of L5178Y/TK+/- cells were pelleted by centrifugation and resuspended in culture medium with 5% heat inactivated horse serum. The resulting cell suspension was diluted to 200.000 cells/mL. In the main experiments with EMS, EMS (0.75 µL/mL) was added to the cell suspension. For this experiments, aliquots without EMS were used as negative control. EMS was used in combination with the test item EUF or the reference item FA in order to enable detection of formaldehyde-specific DNA-protein cross-links. For each treatment 1 mL of the cell suspension was transferred to 1.5 mL reaction cups.
In the course of this study, treatments with and without metabolic activation were carried out. The test item EUF and the reference item FA were both pre-diluted with 10% methanol to generate appropriate stock solutions (500-fold). Two µL of the stock solutions were then added to 1 mL of cell suspension. Control suspensions received 2 µL of 10% methanol. In the main experiments with S9-mix, 50 µL/mL S9-mix were added to the incubation media, being equivalent to 0.6 mg S9-fraction/mL medium. Cultures were incubated for 1 h at 37°C in the dark by horizontal shaking. After the incubation period, 100 µL of the cell suspension were used for electronic determination of the number of viable cells.


NUMBER OF CELLS EVALUATED:
Stained slides were analyzed microscopically using a Comet Assay III software. As a measure of DNA damage, the DNA migration out of the cell nucleus, resembling a comet tail, was analyzed. Head length, head intensity, tail length, tail intensity and the tail moment (% DNA x tail length ), as a metric for DNA migration, were evaluated semi-automatically. 50 nuclei per slide were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: determination of viable cells afetr a 1 hour incubation period with various concentartions

Evaluation criteria:

- Evauation of two slides per treatment, generated by parallel treatment of two independent cell aliquots
- Acceptable staining
- Evaluation of 50 nuclei per slide
- Evaluation of nuclei only in the middle of the slide
- no analysis of overlapping nuclei/comets
- no analysis of comets without heads

Statistics:

Head length, head intensity, tail length, tail intensity and the tail moment (% DNA x tail length ), as a metric for DNA migration, were evaluated semi-automatically. 50 nuclei per slide were evaluated

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Determined by using 9 concentrations of both, EUF and FA. No significant reduction in cell numbers was mediated.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Concerning EUF, concentrations in the main experiments represent a hypothetical FA content of the aqueous solutions, based on FA liberation of about 19.5 % (w/w)

RANGE-FINDING/SCREENING STUDIES: Performed in a prelimonary test on cytotoxicity


ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity found up to a concentration of 2 mM test/reference item in the presence of EMS

Remarks on result:

other: strain/cell type: mouse lymphoma cell line L5178Y/TK+-

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results
positive

The test item EUF showed a positive reaction in the comet assay in L5178Y/TK+/- mouse lymphoma cells. The dose-dependent induction of DNA-protein cross links was comparable to FA (formaldehyde) as reference item at the corresponding concentrations.
Thus, FA seemed to be the main DNA damaging component of EUF(which is a FA releasing mixture).

Executive summary:

The Comet-Assay was conducted following the principles outlined by Tice et al. (2000).

In this study the mouse lymphoma cell line L5178Y/TK^+/-was used.

For cytotoxicity testing and the main experiments, proliferating cell suspensions were pelleted, resuspended in incubation medium and diluted to the desired cell density of 200.000 cells/mL.

For metabolic activation S9-liver homogenate was used. A preliminary range-finding study on cytotoxicity of the test item was performed by evaluating the number of viable cells with various concentrations (2.5 µM, 25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM) of the test item EUF and the reference item FA, each in the presence of the same concentration of EMS (0.75 µL/mL). Five (withEMS) or six (withoutEMS) concentrations of both the test item EUF and the reference item FA were used in the main experiments.

After an incubation period, viable cells were pelleted by centrifugation and slides coated with agarose were prepared. Following electrophoresis, slides were neutralized and DNA was stained with 100 µL ethidium bromide solution.

The stained slides were analyzed. As a measure of DNA damage, the DNA migration out of the cell nucleus, resembling a comet tail, was analyzed. Head length, head intensity, tail length, tail intensity and the tail moment (%DNA x tail length ), as a metric for DNA migration, were evaluated semi-automatically.

The test item EUF showed a positive reaction in the comet assay in L5178Y/TK^+/-mouse lymphoma cells. The dose-dependent induction of DNA-protein cross links was comparable to FA (formaldehyde) as reference item at the corresponding concentrations.
Thus, FA seemed to be the main DNA damaging component of EUF(which is a FA releasing mixture).