2,2'-(ethylenedioxy)diethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-oxydiethanol

EC Number:

203-872-2

EC Name:

2,2'-oxydiethanol

Cas Number:

111-46-6

Molecular formula:

C4H10O3

IUPAC Name:

2,2'-oxydiethanol

Specific details on test material used for the study:

Analyses supplied by the sponsor indicated that the sample contained (by weight) 99.3% diethylene glycol, 0.09% monoethylene glycol, 0.39% triethylene glycol, 0.03% water, and 0.19% unknown components. The specific gravity was 1.1184. For the definitive tests, the test chemical was added directly into the cell culture medium of the test systems. Diethylene glycol is infinitely soluble in water with no solvent incompatibility as indicated in the information supplied by the sponsor. No other analyses of stability or concentration in the test system were performed.

Method

Target gene:

CHO cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

S9 liver homogenate, prepared from Aroclor l254-induced, Sprague-Dawley male rats, as purchased from Microbiological Associates (MBA), Bethesda, MD. The S9 preparation was screened for metabolic activity by the supplier and at BRRC prior to use in our general testing program. Data from MBA showed that the S9 preparation was active with three different activation dependent mutagens in Salmonella bacterial strains TA98 and TA100.

Test concentrations with justification for top dose:

<= 50 mg/mL

Vehicle / solvent:

Test chemical was added directly into the cell culture medium of the test systems.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine and ethylmethanesulfonate

Details on test system and experimental conditions:

Selection of Test Concentrations:
Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would not kill over 90% of the treated cells. Test results wíth diethylene glycol indicated that concentrations up to 50 mg/ml, tested respectively with and without S9 metabolic activation, produced no excessive cytotoxic effects upon cell culture growth or mitotic indices. The 50 mg/ml (5.°G w/v) dose is the maximum dose used in the standard BRRC protocol and it was the highest dose tested with and without S9 activation in this study.


Test Procedures (HPGRT):
At 2 to 3 day intervals after treatment with the test agent, approximately 3 to 5 x 10^5 cells are subcultured in 100 mm tissue culture dishes in F12-D5 medium and incubated at 37°C in a 5 to 6% C02 atmosphere . After a period of at least 7 days to allow "expression" the mutant phenotype, cells of are dissociated with 0.05% to 0.075% trypsin, counted and plated at a concentration of 2 x 10^5 cells/dish in five 100 mm culture dishes (1 x 10^6 total cells) which each contain 10 ml of F12-D5 (TG) selective medium. At this same time interval, cells are diluted and 100 cells/dish are added to four culture plates containing F12-D5 medium (without TG) to assess viability (plating efficiency) of the treated cell population; the value obtained for the viable fraction for cells exposed to various doses and to control agents is used to correct the mutant frequency for cell populations with variable growth ability. All cultures are then incubated for an additional 6 to 8 days to allow growth of cells; medium is then discarded and colonies are fixed and stained for counting. The number of colonies in selection plates and in the viability test are counted by electronic methods, and data are recorded both as mutants/10^6 total cells and mutants/10^6 viable cells

Test Procedures (SCE Assay):
For testing without without metabolic activation, between 2 to 3 x 10^5 cells are plated into 75 cm2 culture flasks in F12-5 medium approximately 40-48 hrs prior to treatment and then incubated at 37°C in a 5 to 6% C02 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and 3 µg/ml BrdU is added to all flasks. Cells are treated with test agents for 5 hrs, media is then removed by suction, cells are rinsed with buffered, physiological salt solution and fresh medium containing 3 µg/ml BrdU is added for at least 24 hrs of additional incubation at 37°C to allow two rounds of cell division. Cells are then harvested and chromosomes are prepared for SCE staining.
Treatment of cells for testing of chemicals which require metabolic activation for mutagenic effectiveness is performed similarly as for treatments without activation, except for three modifications :
1. Approximately 40 to 48 hrs prior to exposure to the test agents, between 2 to 3 x 10^5 cells are added to F12-5 medium in 75 cm2 culture flasks.
2. Before treatment with the test agents, F12-5 medium is removed and F12
medium without serum is added.
3. S9 metabolic activation mixture is added to each flask (including solvent and positive controls) before addition of test agents.
4. Cells are treated for a total of 2 hrs (rather than 5 hrs as in tests without S9) and then incubated for at least 28 additional hours before harvest for chromosome preparation. The exact harvest time can vary depending upon the cytotoxicity of the chemical.

Control Agents:
Positive and solvent control materials were tested concurrently with the test sample to assure the sensitivity of the test system. For the CHO and SCE assays, dimethylnítrosamíne (DMN)-CAS 62-75-9 and ethylmethanesulfonate
(EMS)-CAS 62-50-0 were used as the positive control agents to assure the reliability and sensitivity of the test system for detecting metabolic activation dependent and independent mutagens, respectively. Cell culture medium was used as the vehicle and solvent control agent for this test chemical.

Evaluation criteria:

HPGRT:
The criteria for interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response effect following treatment. When a definite dose-response relationship is not evident but one oi more marginally significant values are obtained, a careful examination of the data from the concurrent positive and negative controls and comparisons to historical control data are used to evaluate the possible biological significance of the responses. Historical negative control data indicate that an average spontaneous mutation frequency in CHO cells of approximately 3 to 4 mutants/10^6 viable cells with a 95 percentile range of 0 to 18 mutants/10^6 viable cells. Statistical comparisons against unusually high or low
spontaneous controls are subjectively scrutinized in respect to the above
expected variability of this test system. Tests are usually repeated to clarify
the biological significance of the data when questionable results are obtained.

For evaluation criteria SCE, see "Any other information on materials and methods incl. tables"

Statistics:

The data were analyzed after transformation of the mutation frequencies (MF) and SCE values according to the conversion method of Box and Cox (1964). For CHO mutation studies with a concurrent control frequency of zero mutants, the variance of recent historical controls was used for the statistical analyses. For SCE data, statistical analyses of historical data at BRRC indicate that an exponent of 0.15 is the appropriate value for transformation of SCE values. Positive controls for the CHO mutation test were run concurrently to assess the sensitivity of the assays in comparison to historical experience with the test system. Data for positive control agents were not compared statistically whenever differences were at least 5 times the concurrent negative control value and results were within the historical positive control range.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: > 50 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The relative cytotoxicity of the various concentrations, tested both in the presence and absence of an S9 metabolic activation system, was determined by measuring the relative growth of treated and control cells incubated overnight following removal of the test chemical. The authors observed that diethylene glycol was not highly cytotoxic when tested either with or without S9 metabolic activation. A concentration of 50 mg/ml produced 16% inhibition of growth without S9 and 27% inhibition with S9. For the definitive tests, a concentration range between 30 to 50 mg/ml was tested in the mutagenicity tests with and without S9. The 50 mg/ml (5% w/v) dose is the usual maximum dose for non-cytotoxic chemicals tested by the BRRC Standard Protocol to avoid possible artefacts produced by non-physiological cell-culture conditions at higher doses.

Applicant's summary and conclusion

Executive summary:

The test substance was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) Mutation test and the Sister Chromatid Exchange (SCE) test. The results indicated that the test substance did not produce a dose-related or repeatable, significant mutagenic effect in either of the in vitro screening tests employed. No detectable cytotoxicity to CHO cells was produced by the test substance even at the maximum concentration of 50 mg/mL (5 % w/v) typically evaluated as the highest dose in these tests following the standard BRRC protocol. The test substance was neither genotoxic nor cytotoxic to CHO cells under the conditions of these two in vitro test systems.