Reaction mass of 2,2'-(Ethylenedioxy)diethanol and 3,6,9-Trioxaundecane-1,11-diol

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:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

in vitro mammalian cell transformation assay

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:

Briefly, the test chemical was analyzed by the sponsor and found to consist of 99.3% diethylene glycol, 0.09% monoethylene glycol, 0.39% triethylene glycol, 0.19% unknown impurities and 0.03% water. The test chemical was added directly into the cell culture medium of the test system without dilution . No analyses of stability or concentration in the medium were performed. Diethylene glycol is infinitely soluble in water and no solvent incompatibilities were listed by the sponsor.

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

Test concentrations with justification for top dose:

30 - 50 mg/ml

Vehicle / solvent:

Cell-culture medium was used as the solvent as the test material was added directly into the cell-culture test system.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: cyclophosphamide and triethylenemelamíne

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 kill, no more than (approximately) 90% of the treated cells. Test concentrations for the SCE test were chosen so that sufficient numbers of cells in the second division (M2) would be available for determination of SCEs. Test results with diethylene glycol indicated that concentrations up to 50 mg/ml, the maximum concentration used for evaluation of test chemicals following the BRRC standard test protocol, were not cytotoxic to CHO cells. For the definitive tests, 50 mg/ml was also selected as the maximum dose and additional concentrations below this dose were evaluated. In this tests, doses between 30 and 50 mg/ml were evaluated for genotoxic potential.
Control Agents: Positive and solvent control materials were tested concurrently with the test sample to assure both the sensitivity of the test systems and the concurrence of the results to historical test performance at BRRC. For this assays, dimethylnitrosamine (DMN)-CAS 62-75-9 and ethylmethanesulfonate (EMS)-CAS 62-50-0 were used as positive control agents to assure the sensitivity and reliability of the test system for detecting metabolic activation dependent and independent mutagens, respectively. Cell culture medium was used as the negative control.
Metabolic Activation: S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats, was purchased- from Microbiological Associates, Bethesda, MD. The S9 preparation used for the CHO test was screened for appropriate metabolic activity by the supplier with three activation-dependent mutagens and Salmonella strains TA98 and TA100. A volume of 50 ul of S9 homogenate was used for each 1.0 ml of the S9 activation mixture.

Evaluation criteria:

For evaluations of direct clastogenic potential, CHO cells were exposed to diethylene glycol and appropriate controls for the complete 12 hour period without S9 activation. Determination of indirect genotoxic potential, requiring metabolic activation by liver S9-homogenate, was studied with a 2-hour exposure period to the test chemical and S9 activation system.

Statistics:

Statistical analyses of the test data employed the Fisher's Exact Test (one-tailed) to determine significance of differences between the test and control populations. This statistical test was considered appropriate for the analysis of the data because it is a distribution independent test and cytogenic data often vary from a normal distribution required for parametric analyses. A positive test result was interpreted by the attainment of differences from the control at the p < 0.05 level of significance for at least one test concentration, together with an indication of a concentration-related effect of exposures or
reproducibility between the duplicate cultures. Rounding of data to either two decimal places or to the appropriate number significant figures was performed for presentation on tables. Although statistically significant decreases in genotoxicity indices can occur because of cytotoxic responses, only statistically significant increases ín responses above control values are indicated on tables for simplicity. All statistical tests were performed to determine whether the treatment with the test agent produced a response statistically different from the value(s) obtained with the concurrent solvent control cultures.

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:

Diethylene glycol did not produce dose-related, statistically significant increases in the incidence of chromosome aberrations in CHO cells in tests conducted with and without the addition of a rat-liver homogenate, S9 metabolic activation system. A single statistical indication of an increase above control values was obtained for the lowest dose sampled at 12 hr after test initiation in the test with S9 activation. This result was not repeated at higher doses and the level of the increase was essentially the same as the variation in the concurrent negative control values. For these reasons, the statistical indication was not considered biologically relevant. The positive control agents, cyclophosphamide and triethylenemelamine, both produced significant increases in chromosome aberrations indicative of the appropriate, reliability of the test system. The control cultures employing only the culture growth medium had low and acceptable levels of chromosome aberrations typical for these cultured cells. Diethylene glycol was concluded to lack significant genotoxic activity under the conditions employed for this in vitro test system.

Any other information on results incl. tables

No statistically significant increase in the number of SCEs was observed with any of the test cultures treated with the test agent. The incidence of SCEs in all of the cultures exposed to the test agent was within the historical negative control range for this test at BRRC (Methods, Section G1). Diethylene glycol was considered inactive under the non-activation conditions of this sensitive in vitro assay.

Table 1: Preliminary cytotoxicity test for determination of appropriate dose range for subsequent genotoxicity tests

Test chemical [mg/mL]	Initial cell concentration* [cells/plate x 10^5]	Final cell concentration** [cells/plate x 10^5]		Percentage relative to solvent control [%]
		Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
0.0 (solvent-medium) 30 35 40 45 50	5.0 5.0 5.0 5.0 5.0 5.0	19.76 20.42 20.42 18.79 16.15 19.84	21.57 18.86 20.79 20.50 21.24 22.43	100.0 103.3 103.3 95.1 81.7 100.4	100.0 87.4 96.4 95.0 98.5 104.0

*5 x 10⁵CHO cells were inoculated uniformly into all culture flasks approximately 24 h prior to chemical treatments

** determined approximately 20 to 24 h after removal of the test agents

Table 2: Cytotoxicity testing summary - mitotic indices (percentage of dividing cells) after an 8 h treatment

Test chemical [mg/mL]	No. of cells counted	No. of cells in metaphase	% cells in metaphase	% of control
Test without S9 activation system
0.0 30 35 40 45 50	500 500 500 500 500 500	10 10 9 10 10 11	2.0 2.0 1.9 2.0 2.0 2.2	100.0 100.0 90.0 100.0 100.0 110.0
Test with S9 activation system
0.0 30 35 40 45 50	500 500 500 500 500 500	9 9 10 8 13 11	1.9 1.9 2.0 1.6 2.5 2.2	100.0 100.0 111.1 88.9 144.4 122.2

Table 3: Summary of combined test data and statistical analyses

Test chemical [mg/mL]	Total cells with aberrations	Total cells scored	Percentage aberrant cells (± SD)	Level of statistical* difference above solvent control value	Total cells with aberrations	Total cells scored	Percentage aberrants cells (± SD)	Level of statistical* difference above solvent control value
Test without S9 activation system
	8 h sampling time				12 h sampling time
0.0 30 40 50 TEM 1.0 µg/mL	2 3 7 3 18	100 100 100 100 50	2.0 (0.0) 3.0 (4.2) 7.0 (4.2) 3.0 (1.4) 36.0	- NS NS NS c	2 2 4 2 -	100 100 100 100 -	2.2 (2.8) 2.0 (2.8) 4.0 (0.0) 2.2 (0.0) -	- NS NS NS -
Test with S9 activation system
	8 h sampling time				12 h sampling time
0.0 30 40 50 Cyclophosphamide 15 µg/mL	6 4 1 6 9	100 100 100 100 50	6.0 (0.0) 4.0 (0.0) 1.0 (1.4) 6.0 (0.0) 18	- NS NS NS a	1 7 4 5 -	100 100 100 100 -	1.0 (1.4) 7.0 (1.4) 4.0 (2.8) 5.0 (1.4) -	- a NS NS -

*significance based on analyses using Fisher’s Exact Test, 1-tailed: a = 0.05 > p > 0.01; c = p < 0.001; NS = p > 0.05

Applicant's summary and conclusion

Conclusions:

The test substance did not produce significant, dose-related effects in the CHO in vitro chromosome aberration assay when tested with and without addition of an S9 metabolic activation. The results indicate that the test substance does not possess significant clastogenic or cytotoxic potential under the conditions of this in vitro test system.

Executive summary:

The test substance was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) in vitro chromosome aberration test system. Test concentrations of diethylene glycol ranged from 10 mg/mL to 50 mg/mL in tests both with and without a rat-liver S9 metabolic activation system. Test concentration ranges were chosen on the basis of a preliminary cytotoxicity test. The highest three doses which did not produce excessive cytotoxic inhibition of mitotic cells were scored for incidences of chromosome aberrations.

Results obtained in tests with and without a rat-liver homogenate, S9 metabolic activation system, indicated that the test substance did not produce significant, dose-related increases in chromosome aberrations in comparison to values of control cultures. No evidence of clastogenic or cytotoxic effects were observed under the conditions of this test system up to the maximum 50 mg/mL dose used in this test following the standard BRRC protocol.