Reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: genome mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

20 January - 31 July 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Reference 2

Reference Type:

publication

Title:

Genotoxicity of tetraethylene glycol (TEG) evaluated with multiple in vitro and in vivo assays

Author:

Slesinski, R.S. et al.,

Year:

1989

Bibliographic source:

Toxicologist 9, 232

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency Health Effects Test Guidelines. HG-Gene Muta-Somatic cells, EPA Report No. 560/6-83-001, October 1983.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

3,6,9-trioxaundecane-1,11-diol

EC Number:

203-989-9

EC Name:

3,6,9-trioxaundecane-1,11-diol

Cas Number:

112-60-7

Molecular formula:

C8H18O5

IUPAC Name:

2,2'-[oxybis(ethane-2,1-diyloxy)]diethanol

Test material form:

other: Water-clear, slightly to nonviscous liquid.

Details on test material:

A sample of tetraethylene glycol was received for testing on January 16, 1986 from Union Carbide Corporation, Texas City, Texas. Gas chromatographic analysis performed by the sponsor indicated that the sample was approximately 99.69% pure and contained 0.056% triethylene glycol and 0.058% pentaethylene glycol.

A second sample of tetraethylene glycol was received on July 1, 1986 from Union Carbide Corporation, Texas City, Texas. Gas chromatographic analysis performed by the sponsor indicated that this sample contained 99.941% tetraethylene glycol. The major impurities are triethylene glycol at 0.028% and pentaethylene glycol at 0.012%..

Method

Target gene:

HGPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) cells were obtained from Abraham Hsie at Oak Ridge National Laboratory with the designation CH0-Kl-BH4-(subclone D1) (referred to simply as CHO for report purposes).

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver S9 homogenate (prepared from Aroclor 1254 induced, Sprague-Dawley, male rats) is purchased from Hazleton Biotechnologies, Kensington, MD or Microbiological Associates, Bethesda, MD.

Test concentrations with justification for top dose:

0, 24, 29, 35, 42 and 50 mg/ml

Vehicle / solvent:

Test material was used neat.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

other: Cell culture medium was used for control samples

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine and ethylmethanesulfonate

Details on test system and experimental conditions:

CHO Mutation Test
Dose Selection - Appropriate concentrations for mutagenicity testing were determined by preliminary measurements of cytotoxicity to CHO cells of a range of concentrations tested both in the presence and absence of a rat-liver S9 metabolic activation system. Selection of a suitable range of concentrations for testing was based upon an estimate of the doses which would not produce excessive cytotoxicity to the treated cells. A dose of 50 mg/ml is the BRRC limit for the highest dose evaluated in testing freely-soluble, noncytotoxic chemicals in this test system. Cell-culture medium was used as the solvent for dilutions. All dilutions were prepared immediately prior to testing.

Test Procedure - Duplicate cultures of CHO cells were exposed for 5 hours to a minimum of five concentrations of tetraethylene glycol in tests both with and without the addition of a rat-liver S9 metabolic activation system. Various dose levels of tetraethylene glycol for testing were attained by direct addition of various aliquots of the undiluted test agent into the cell culture medium. The surviving fraction was determined at 18 to 24 hours after the removal of the test chemical using 4 plates/culture and 100 cells/plate. The mutant fraction was determined after a 9 to 12 day sub-culturing period to allow "expression" of the mutant phenotype. The mutant fraction was assessed in selective medium with 2 x 10(5) cells/plate in 5 plates/dosed culture (i.e. 1 x 10(6) total cells/dosed culture). The plating efficiency of these cells was assessed in non-selective medium using 4 plates/dosed culture with 100 cells/plate.

The mutagenicity/survival/plating efficiency data from at least the top five concentrations which allowed sufficient cell survival for assessment of survival and quantification of mutants are typically presented in the tables. The percentage of cells surviving the treatment, the numbers of mutant colonies, the percentage of clonable cells and the calculated number of mutants/10(6) clonable cells are presented in tabular form.

SCE Test
Dose Selection - Selection of a suitable range of doses for testing was based either upon cytotoxicity data obtained as part of the CHO mutation test or from preliminary experiments to determine cytotoxicity of the test chemical. For freely-soluble noncytotoxic chemicals, a maximum concentration of 50 mg/ml is used for this test system at BRRC to decrease the possibility of artefacts resulting from nonphysiological conditions in the cell-culture system.

Test Procedure - Production of SCEs following exposure to various concentrations of tetraethylene glycol was studied with duplicate cultures of CHO cells tested both with and without the incorporation of a rat-liver S9 metabolic activation system. Various concentrations of tetraethylene glycol for testing were attained by direct addition of various aliquots of the undiluted test agent into the culture medium.

For determination of direct genotoxic action, CHO cells were exposed to tetraethylene glycol and appropriate controls for 5 hours without S9 activation. Indirect activity, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU), required to differentiate between the individual "sister" chromatids by SCE staining, was present at a concentration of 3 ug/ml in the growth medium during treatment and during the culture period following exposure. A total of twenty-five cells/concentration was examined for SCE frequencies using duplicate cultures. At least 5 dose levels were tested both with and without metabolic activation. SCE production was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division. The number of SCEs/cell, mean number of SCEs/chromosome and the level of statistical significance of the increases above the concurrent solvent control values are presented in tabular form.

Osmolality - Published research has shown that conditions of high osmotic pressure can induce weak levels of chromosome damage. To assess the osmolality of each of the dose levels tested, various aliquots of the undiluted test sample were added directly to tissue culture medium to achieve the concentrations tested in the definitive chromosome aberration experiments. Osmolality was measured with an Advanced CRY0MATIC OSMOMETER (model 3 CII). Osmolality was assessed for culture conditions both with and without the presence of the S9 metabolic activation system. The osmolality of the culture medium alone was assessed as a reference point for evaluating the effects of the various concentrations of the test article.

Control Agents
Positive, negative 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 the CHO and SCE 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 for statistical comparisons.

Metabolic Activation
S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley M A L E RATS, was purchased from Microbiological Associates, Bethesda, MD. The S9 preparation used for the CHO gene mutation test was found to have significant metabolic activity with three activation dependent positive control agents, tested for mutagenic activity by the supplier, using Salmonella bacterial strains TA98 and TA100. Following screening tests on this lot of S9 at BRRC a volume of 50 ul of S9 homogenate was found to be a suitable S9 concentration per milliliter of the S9 activation system.

For the two SCE tests, another lot of S9 homogenate, purchased from Litton Bionetics, Kensington, MD, contained 40 mg/ml protein and had a
benzo[cc]pyrene-hydroxylase activity of 15 nmol hydroxybenzpyrene/20 min/mg protein (assayed by Litton); a final concentration of 600 ug of S9 protein was used per 1.0 ml of the S9 activation mixture.

Typically 1.0 ml of the complete metabolic activation system (S9 homogenate plus cofactors) was added per each 4.0 ml of culture medium.

Evaluation criteria:

Evaluation of results from in vitro mutagenicity tests must take into consideration a comparison of both concurrent and historical control data for interpretation of the biological significance of the results. Each test system has been found to vary both within and between laboratories and evaluations only against concurrent controls may be misleading. The range of variability of negative control data for the tests at BRRC, updated as of December 31, 1985, is provided for comparative purposes. Because the data from many systems do not follow a normal distribution, the mean and median are both given as well as the 95 percentile range for typical test variability.

1. CHO mutation test
Negative controls: n = 30
mean = 4.0 mutants/10(6) clonable cells
S.D. = 7.0
median = 1.2 mutants/10(6) clonable cells
95 percentile range = 0 to 25.6 mutants/10(6) clonable cells
2. SCE test
Negative controls: n = 30
mean = 0.509 SCEs/chromosome
S.D. = 0.087
median = 0.498 SCEs/chromosome
95 percentile range = 0.227 to 0.666 SCEs/chromosome

Statistics:

Data from the SCE and CHO tests do not follow a normal distribution according to experience with historical controls. Thus, the data were analyzed after transformation of the mutation frequencies (MF) and SCE values according to the conversion method of Box and Cox (1964). This procedure for CHO data follows procedures described by Snee and Irr: (MF + l )( 0.15) (Snee, R.D. and J.D. Irr, Mutation Research, 85 (1981), 77-93). 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 (BRRC Intramural Report 46-64). 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

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

HGPRT

Cytotoxicity / choice of top concentrations:

cytotoxicity

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

SCE

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

CHO MUTATION TEST
Selection of Test Concentrations
In a preliminary study, CHO cells were exposed for five hours to a range of concentrations from 0.003 to 50 mg/ml of the test material to
determine an appropriate cytotoxic range of doses. 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. We observed that tetraethylene glycol was not remarkably cytotoxic to CHO cells even at 50 mg/ml, the highest dose generally used for this assay system at BRRC. Higher doses of tetraethylene glycol and other test chemicals are not tested because of the inherent potential for nonphysiological effects caused by the extremely high osmotic conditions of the cell culture medium.

For the definitive tests, a concentration range between 24 to 50 mg/ml was tested in the mutagenicity test without S9 (Tables 1 and 2) and in the presence of S9 (Tables 3 and 4.

Determination of Mutation Induction
Survival (Cytotoxicity)
Tables 1 and 3 present the cytotoxicity data determined by the plating efficiency of cells seeded into cloning plates at approximately 24 hours post-exposure to tetraethylene glycol.The test chemical produced dose-related cytotoxicity to CHO cells treated both with (Table 4) and without (Table 2) S9 metabolic activation. The decrease in plating efficiency of CHO cells indicates that biologically effective doses were tested and this likely is an underestimate of the cytotoxicity because of the 18 hr recovery period post-exposure.

Mutation
Tables 2 and 4 present the data for production of mutants by the test chemical and control agents. Tetraethylene glycol did not produce a dose-related increase in the number of mutants/106 viable cells over the range of concentrations tested either with or without the presence of an S9 metabolic activation system. No concentration of the test agent produced an increase in the incidence of mutations which was statistically different from the concurrent solvent control in the test with or without S9 activation. Small numerical increases in the mutant fraction obtained with some concentrations in both tests were within the historical control range of variability for this test system at BRRC. Also, these values were not statistically different from the concurrent control. Tetraethylene glycol was not mutagenic to CHO cells in the tests performed with and without metabolic activation.

Mutation values for the solvent controls for tests both with and without S9 activation were in an acceptable and low range based upon the variability for this test system experienced with historical control values at BRRC. Quantitative increases in the numbers of mutations of at least 5 to 10-fold greater than the concurrent controls were obtained for the DMN and EMS positive controls in all experiments and these values were within the expected range of values observed in previous experiments with this test system at BRRC. Positive control data were not compared statistically because of the obvious positive effects. Statistical comparisons to concurrent control values of zero were performed by using the variance values for the historical control data for this test at BRRC.

SCE TEST
Selection of Test Concentrations
In preliminary cytotoxicity tests, a concentration of 50 mg/ml produced 18.6% inhibition of culture growth when tested with an S9 metabolic activation system. The 50 mg/ml dose tested without metabolic activation produced approximately 21% growth inhibition.

For the definitive tests to determine potential effects upon SCEs, a range of doses from 24 mg/ml to 50 mg/ml was tested with and without addition of an S9 metabolic activation system. The highest three concentrations which permitted a suitably high mitotic index were examined for SCEs. A 50 mg/ml dose is the highest concentration evaluated in the test system at BRRC as possible artifacts may be caused by excessively high osmotic concentrations in in vitro chromosome tests (Galloway et. al. 1985, Env. Mutagenesis 7, Suppl. 3, pp 48-49).

Determinations of Effects upon SCEs
1. The data for SCE production in CHO cells treated with various dose levels of tetraethylene glycol or with positive, negative or solvent control agents without an S9 metabolic activation system are summarized in Table 5. A statistically significant increase in the number of SCEs was observed with all three dose levels of the test agent evaluated for SCEs. The magnitude of the increases was not remarkably high in comparison to positive control agents used for this test. Also, no definite dose-related trend was apparent in the data. The indication of consistent statistically significant differences from the control values indicated that the test result should be considered a weakly positive effect. However, a repeat test was considered necessary to determine the reproducibility of the SCE effects and to rule out the possibility of contaminants in the test sample.

The number of SCEs produced by the concurrent EMS positive control was highly statistically different from the values for the concurrent solvent controls. These data indicated an appropriate sensitivity of the test system comparable to our historical positive control data. The number of SCEs obtained with the solvent and medium controls were also in an acceptable range of values included in the variability encountered in our historical control values for this test at BRRC.

2. SCE values obtained following treatments of CHO cells with tetraethylene glycol in the presence of an S9 metabolic activation system are presented in Table 6. A statistically significant increase in the SCE values was produced with each of the three doses of the test agent evaluated for SCEs, in comparison to the concurrent controlin the presence of S9 activity. The magnitude of the increases and the absence of a distinct dose-related trend in the values was similar to the data obtained without S9 activation. Although the relative level of the SCE increases were low, the statistical indication of a significant difference above control values was used to consider the test as a positive indication of weak genotoxic activity. The biological significance of such low levels of activity at relatively high exposure concentrations must be evaluated with caution, both with regard to the possibility of contaminants in the test chemical and reproducibility of the positive effects.

The SCE values for the negative and solvent controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCEs were produced by the DMN positive control which indicated that the metabolic activation system was suitably active.

3. In the data from the test without S9, the highest dose (50 mg/ml) produced excessive inhibition of the numbers of cells in mitosis and it could not be scored. The remaining lower doses did not produce cell-cycle delays evident in this assessment.

In the test with S9 activation, no observable cell cycle delays were evident in the proportion of cells in the first and second mitotic division. The highest dose of 50 mg/ml was less cytotoxic than in the test without S9 and this dose was scored in the test.

The cytotoxicity data obtained from this test demonstrated that the doses evaluated for SCEs did not produce cell-cycle delays despite the use of extremely high concentrations.

SECTION III - SCE - Repeat Test
Purpose of Repeat Test
An initial sister chromatid exchange test on tetraethylene glycol (BRRC Sample No. 49-8) indicated a weak, but consistent, statistically significant effect upon the incidence of SCEs in CHO cells. This result was not considered sufficient for an unequivocal determination for two reasons: 1) the elevation in SCEs did not demonstrate a definite dose-related trend which is characteristic for positive genotoxic agents in this test system; and 2) analyses of the test chemical performed by the sponsor indicated that it contained higher levels of unknown impurities relative to the typical specifications for this material. A repeat test with a second sample of tetraethylene glycol with acceptable purity specifications was conducted to evaluate the reproducibility of the indication of genotoxic potential in the initial SCE test.

Determinations of Effects upon SCEs
1.The data for SCE production in CHO cells treated with various dose levels of tetraethylene glycol or with positive, negative or solvent control agents without an S9 metabolic activation system are summarized in Table 7.. A statistically significant increase in the number of SCEs was observed for all of the highest three test doses evaluated for SCEs. However, only the highest concentration evaluated (42 mg/ml) produced a consistent effect with both of the cell cultures treated with the test chemical. The lack of a definite dose-response trend in the SCE data was consistent with the results from the previous test on the first sample of tetraethylene glycol. The test result was considered to be a positive indication of genotoxic potential based upon the statistical indications of an increase over control values with three of the test doses evaluated. However, the biological significance of such weak, non-dose-related effects at high osmotic concentrations of the test agent should be evaluated with caution.

2. SCE values obtained following treatments of CHO cells with tetraethylene glycol in the presence of an S9 metabolic activation system are presented in Table 8. A statistically significant increase in the SCE values was produced with each of the three doses of the test agent evaluated for SCEs, in comparison to the concurrent control. The magnitude of the increases and the absence of a distinct dose-related trend in the values was similar to the data obtained without S9 activation. Although the relative level of the SCE increases were low, the statistical indication of a significant difference above control values was used to consider the test as a positive indication of weak genotoxic activity. The biological significance of such low levels of activity at relatively high exposure concentrations must be evaluated with caution, both with regard to the possibility of contaminants in the test chemical and reproducibility of the positive effects.

The test chemical was slightly less cytotoxic in the presence of the rat-liver activation mixture than observed in the test without activation. The data in this test showed similar statistical indications of a positive effect, low SCE increases, absence of clear dose-response trends and lack of reproducibility for duplicate cultures as those observed in the test without S9 activation. The reservations expressed in the previous section regarding the cautions interpretation of weak effects at extremely high doses must be reiterated for this test result. However, because of the statistically positive increases above the concurrent controls, the test was concluded to be a positive indication of weak genotoxic activity.

The SCE values for the negative controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCEs were produced by the DMN positive control which indicated that the metabolic activation system was suitably active.

3. In the data from the tests with and without S9, no remarkable increase in cells at the first mitotic division was observed on the slides evaluated for SCEs. The cytotoxicity data obtained from this test demonstrated that the doses evaluated for SCEs did not produce an adverse effect upon the progression of the cell population through the mitotic cycle.

Determination of the Osmolality of the Test Concentrations.
Nonphysiological conditions of high osmolality (>/= 450 mOsm/Kg H20) have been shown to produce significant levels of chromosome damage (Deasy et al. 1986; Galloway et al. 1985). The osmolality of the tissue culture medium and the various concentrations of the test article in the tissue culture medium tested was measured both in the presence and absence of an S9 metabolic activation system. Results are expressed in milliosmoles/Kg of H2O. The osmolality of the tissue culture medium used for the respective test exposures was 288 in the presence of S9 (minum serum) and 295 in the absence of S9 (plus 5% serum). The osmolality of the test concentrations of the test article which ranged from 423 mOsm/Kg H20 to 605 mOsm/Kg H20. The osmolality was similar for concentrations of each dose level prepared with or without S9. The highest concentration tested (50 mg/ml) had an osmotic strength that was twice that of the culture medium alone and all five concentrations of tetraethylene glycol that were tested exceeded 400 mOsm/Kg H20. In previous tests conducted at BRRC on a group of related glycols, no positive effects upon SCEs were noted with ethylene, diethylene and triethylene glycol within a similar or higher range of osmolalities. Thus, the absence of a direct relationship between SCE induction and osmolality for these related chemicals indicated that the results with tetraethylene glycol in this study in tests performed with and without S9 activation should be considered evidence for weak genotoxic activity.

Remarks on result:

other: slight, but statistically significant and repeatable increases in SCEs; not dose responsive

Any other information on results incl. tables

Table 1 Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Cytotoxicity After Chemical Treatment Without Metabolic Activation

Test Material	Total Number of Colonies	% Survival Mean (+/-SD)	% of Combined Solvent Controls
Tetraethylene Glycol, mg/ml	Test Without S9 Activation
24A	399	99.8 (7.9)	101.3
24B	388	97.0 (4.1)	98.5
29A	346	86.5 (7.4)	87.8
29B	334	83.5 (2.9)	84.8
35A	363	90.8 (11.1)	92.1
35B	383	95.8 (16.6)	97.2
42A	284	71.0 (8.4)	72.1
42B	270	67.5 (1.3)	68.5
50A	214	53.5 (11.7)	54.3
50B	225	56.2 (9.7)	57.1
Controls (Cell-culture medium)	389	97.2 (5.9)	98.7
Controls (Cell-culture medium)	399	99.8 (17.9)	101.3
Positive Control (EMS, 200 ug/ml)	393	98.2 (11.0)	99.7

*100 cells inoculated into each plate after approximately an 18 hr recovery period following removal of the test chemicals.

EMS - ethylmethanesulfonate.

Table 2 Chinese Hamster Ovary (CHO) Mutation Assay; Test Without Metabolic Activation System Results on Evaluation of Plating Efficiency and Mutant Frequencies Determined After Expression Period

			Mutant Colonies		Corrected***Mutation
Test Material	Mean Colonies/Plate (+ S.D.)	% of CombinedSolvent Controls	Mean (+ S.D.)	Total Colonies**	Frequency (x10 ^-6)
Tetraethylene glycol, mg/ml	Tested Without S9 Activation
24A	104.2 (3.4)	94.9	1.0 (0.7)	5	4.8
24B	122.0 (28.5)	111.2	0.8 (1.1)	4	3.3
29A	110.8 (8.8)	101.0	0	0	0
29B	91.5 (13.4)	83.4	0.2 (0.4)	1	1.1
35A	126.0 (25.1)	114.8	0.6 (0.9)	3	2.4
35B	92.8 (14.1)	84.6	0.2 (0.4)	1	1.1
42A	126.5 (18.8)	115.3	0	0	0
42B	113.8 (18.6)	103.7	1.8 (1.3)	9	7.9
50A	103.5 (11.5)	94.3	0	0	0
50B	93.8 (15.8)	85.5	0.6 (0.9)	3	3.2
Control (Cell-culture medium)	122.0 (25.3)	111.2	0	0	0
Control (Cell-culture medium)	97.5 (21.2)	88.8	0	0	0
Positive control (EMS, 200 ug/ml)	106.0 (24.1)	96.6	29.6 (4.9)	148	139.6

** 2 x 10⁵ cells inoculated in each of 5 plates, (1 x 10⁶ total cells),

***Mutants/10⁶ clonable cells: total # mutant colonies divided by viable fraction;

Statistical difference above control: a = 0.05 > p > 0.01; b = 0.01 > p > 0.001; c = p < 0.001.

No superscript indicates p > 0.05. Data for test chemical effects were analyzed by Method of Irr and Snee; data for positive controls were compared to historical ranges but were not analyzed statistically.

Table 3 Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Cytotoxicity 24 Hours After Chemical Treatment With Metabolic Activation

Test Material	Total Number of Colonies	% Survival Mean (+ S.D.)	% of Combined Solvent Controls
Tetraethylene glycol, mg/ml	Test With S9 Activation
24A	462	115.5 (19.8)	95.5
24B	473	118.2 (23.8)	97.7
29A	445	111.2 (17.6)	91.9
29B	421	105.2 (5.7)	87.0
35A	466	116.5 (31.4)	96.3
35B	366	91.5 (7.8)	75.6
42A	390	97.5 (23.8)	80.6
42B	396	99.0 (14.7)	81.8
50A	257	64.2 (5.1)	53.1
50B	266	66.5 (11.7)	55.0
Control (Cell-culture medium)	485	121.2 (16.4)	100.2
Control (Cell-culture medium)	483	120.8 (26.5)	99.8
Positive Control (DMN, 100 mg/ml)	174	43.5 (4.7)	36.0

DMN - dimethylnitrosamine

Table 4 Chinese Hamster Ovary (CHO) Mutation Assay; Test With Metabolic Activation System Results on Evaluation of Plating Efficiency and Mutant Frequencies Determined After Expression Period

	Plating Efficiency		Mutant Colonies		Corrected*** Mutation
Test Material	Mean Colonies/Plate (+ S.D.)	% of Combined Solvent Controls	Mean (+ S.D.)	Total Colonies**	Frequency (x10 ^-6)
Tetraethylene glycol, mg/ml	Tested With S9 Activation
24A	96.0 (20.8)	90.0	0	0	0
24B	106.2 (15.5)	99.5	0.4 (0.5)	2	1.9
29A	91.8 (17.8)	86.0	2.2 (0.8)	11	12.0
29B	96.5 (20.1)	90.4	0.4 (0.5)	2	2.1
35A	101.2 (13.9)	94.8	0.2 (0.4)	1	1.0
35B	113.0 (20.8)	105.9	1.4 (1.3)	7	6.2
42A	102.5 (9.8)	96.1	1.2 (1.1)	6	5.9
42B	102.2 (10.6)	95.8	0	0	0
50A	115.2 (14.7)	108.0	0.4 (0.9)	2	1.7
50B	104.8 (4.2)	98.2	0	0	0
Control (Cell-culture medium)	103.2 (22.2)	96.7	1.6 (0.9)	8	7.7
Control (Cell-culture medium)	110.2 (26.0)	103.3	0	0	0
Positive control (DMN, 100 ug/ml)	88.0 (26.6)	82.5	31.2 (5.1)	156	177.3

** 2 x 10⁵ cells inoculated in each of 5 plates, (1 x 10⁶ total cells).

***Mutants/10⁶ clonable cells: total # mutant colonies divided by viable fraction. Statistical difference above control: a = 0.05 > p > 0.01; b = 0.01 > p > 0.001; c = p < 0.001. No superscript indicates p > 0.05. Data for test chemical effects were analyzed by Method of Irr and Snee; data for positive controls were compared to historical ranges but were not analyzed statistically.

Table 5 Sister Chromatid Exchange (SCE) Assay; Production of SCEs by Tetraethylene Glycol Tested Without S9 Metabolic Activation - 5-Hour Treatment

Test Material	Total # of	Total # of		Mean Number of SCEs/	Significance Above
Tetraethylene glycol, mg/ml	Chromosomes	SCEs	SCEs/Cell*	Chromosome** (+ S.D.)	Solvent Controls***
29A	498	363	14.5	0.73 (0.25)	c
29B	501	355	14.2	0.71 (0.12)	c
35A	496	369	14.8	0.74 (0.29)	c
35B	500	411	16.4	0.82 (0.23)	c
42A	497	371	14.8	0.75 (0.24)	c
42B	496	418	16.7	0.84 (0.19)	c
Control (Cell-culture medium)	499	232	9.3	0.46 (0.14)	-
Control (Cell-culture medium)	501	243	9.7	0.49 (0.15)	-
Positive Control (EMS, 100 ug/ml)	500	673	26.9	1.35 (0.25)	c

* Twenty-five cells examined per dosed culture.

** Mean value of SCE/chromosome determined from the values of the individual cells examined.

***Statistical significance above solvent control: c: p < 0.001.

Data analyzed by Student's t-test by comparing individual test groups with the combined solvent control groups. Significances noted in parentheses are the combined test groups compared statistically against the combined solvent controls.

EMS - ethylmethanesulfonate

Table 6 Sister Chromatid Exchange (SCE) Assay: Production of SCEs by Tetraethylene Glycol Tested With S9 Metabolic Activation - 2-Hour Treatment

Test Material	Total # of	Total # of		Mean Num ber of SCEs/	Significance Above
Tetraethylene glycol, mg/ml	Chromosomes	SCEs	SCEs/Cell*	Chromosomes** (+S.D.)	Solvent Controls***
35A	501	342	13.7	0.68 (0.20)	c
35B	502	384	15.4	0.76 (0.22)	c
42A	500	384	15.4	0.77 (0.30)	c
42B	499	381	15.2	0.76 (0.18)	c
50A	497	405	16.2	0.82 (0.28)	c
50B	504	374	15.0	0.74 (0.23)	c
Control (Cell-culture medium)	499	248	9.9	0.50 (0.14)	-
Control (Cell-culture medium)	501	248	9.9	0.50 (0.11)	-
Positive Control (DMN, 300 ug/ml)	499	889	35.6	1.78 (0.53)	c

* Twenty-five cells examined per dose level.

** Mean value of SCE/chromosome determined from the values of the individual cells examined.

*** Statistical significance above solvent control: c: p < 0.001.

Data analyzed by Student's t-test by comparing individual test groups with the combined solvent control groups. Significances noted in parentheses are the combined test groups compared statistically against the combined solvent controls.

DMN-Dimethylnitrosamine

Table 7 Sister Chromatid Exchange (SCE) Assay: Production of SCEs by Tetraethylene Glycol Tested Without S9 Metabolic Activation - 5-Hour Treatment

Test Material	Total # of	Total # of		Mean Number SCEs/	Significance Above
Tetraethylene glycol, mg/ml	Chromosomes	SCEs	SCEs/Cell*	Chromosome** (+S.D.)	Solvent Controls***
29A	496	281	11.2	0.56 (0.18)	NS
29B	498	353	14.1	0.71 (0.14)	c
35A	497	346	13.8	0.70 (0.23)	c
35B	499	283	11.3	0.57 (0.19)	NS
42A	497	327	13.1	0.66 (0.17)	c
42B	499	330	13.2	0.66 (0.16)	c
50	cytotoxic - too few mitotic cells were available to score
Control (Cell-culture medium)	501	235	9.4	0.47 (0.09)
Control (Cell-culture medium)	499	243	9.7	0.49 (0.17)
Positive Control (EMS, 100 ug/ml)	504	644	25.8	1.28 (0.23)	c

* Twenty-five cells examined per dosed culture.

** Mean value of SCE/chromosome determined from the values of the individual cells examined.

***Statistical significance above solvent control: c: p < 0.001; NS: p > 0.05.

Data analyzed by Student's t-test by comparing individual test groups with the combined solvent control groups. Significances noted in parentheses are the combined test groups compared statistically with the combined solvent controls.

EMS - ethylmethanesulfonate

Table 8 Sister Chromatid Exchange (SCE) Assay: Production of SCEs by Tetraethylene Glycol Tested With S9 Metabolic Activation - 2-Hour Treatment

Test Material	Total # of	Total # of		Mean Number of SCEs/	Significance Above
Tetraethyene glycol, mg/ml	Chromosomes	SCEs	SCEs/Cell*	Chromosome** (+S.D.)	Solvent Controls***
35A	503	356	14.2	0.71 (0.22)	c
35B	496	312	12.5	0.63 (0.17)	NS
42A	498	287	11.5	0.58 (0.16)	NS
42B	498	346	13.8	0.70 (0.21)	b
50A	498	360	14.4	0.72 (0.24)	c
50B	494	363	14.5	0.74 (0.31)	c
Control (Cell-culture medium)	498	259	10.4	0.52 (0.14)
Control (Cell-culture medium)	489	248	9.9	0.51 (0.17)
Positive Control (DMN, 300 ug/ml)	495	1339	53.6	2.70 (0.94)	c

* Twenty-five cells examined per dose level.

** Mean value of SCE/chromosome determined from the values of the individual cells examined.

*** Statistical significance above solvent control: b: 0.01 > p > 0.001; c: p < 0.001; NS: p > 0.05.

Data analyzed by Student's t-test by comparing individual test groups with the combined solvent control groups. Significances noted in parentheses are the combined test groups analyzed against the combined, solvent controls.

DMN-Dimethylnitrosamine

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous SCE
negative HGPRT forward mutation assay

Tetraethylene glycol produced weak but statistically significant increases in the incidence of SCEs over the range of concentrations tested with or
without addition of an active S9 metabolic activation system. However, no definite, dose-related effects of exposure on the incidence of SCEs were
evident. Although the biological significance of the results must be evaluated with caution because of the high range of osmotic concentrations evaluated, tetraethylene glycol produced reproducibly positive increases in the incidence of SCEs in CHO cells with the relatively high range of exposure concentrations used for these studies.

Executive summary:

Tetraethylene glycol was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) gene mutation test and the sister chromatid exchange (SCE) test. The results indicated that tetraethylene glycol did not produce a dose-related or repeatable, significant mutagenic effect in the CHO gene mutation assay in the tests with and without S9 activation. The lack of gene mutation activity for tetraethylene glycol was consistent with the lack of mutagenic effects in a previous Ames (Salmonella) gene mutation test

conducted previously (BRRC Project Report 49-59).

Results from a sister chromatid exchange assay indicated that tetraethylene glycol produced statistically significant increases of SCEs in tests with and without rat-liver S9 activation, but the increases did not occur with a dose-related trend characteristic of genotoxic agents. The reproducibility of these equivocal effects was evaluated with a second sample of tetraethylene glycol, because analyses performed by the sponsor revealed an atypical concentration of unidentified impurities in the first sample. The repeat SCE test on a sample of acceptable purity produced essentially identical increases in the incidence of SCEs as obtained with the first test. This weakly positive increase in SCEs was consistent with results in chromosome aberration tests with CHO cells conducted on the first sample of tetraethylene glycol at BRRC (BRRC Project Report 49-90).

The biological significance of weak effects at very high test concentrations must be interpreted with caution because of the possibility of spurious effects caused by the significant alteration of the osmotic strength of the cell-culture medium at these high test concentrations. Conditions of high osmolality (> 450 mOsm/Kg H₂O) produced by noncytotoxic chemicals such as tetraethylene glycol can produce increased incidences of sister chromatid exchanges and chromosome aberrations (Galloway et al. 1985; Deasy et al. 1986). The osmolality of the test concentrations of tetraethylene glycol used for this study was determined to be in the approximate range of 423 to 605 mOsm/Kg H₂O in comparison to 288 to 295 mOsm/Kg H₂O for the cell culture medium alone.

Thus, the possibility that the weak effects observed in this study were the result of a minor contaminant or caused by a physiological artifact cannot be excluded. However, in previous studies on related glycols (e.g. ethylene-, diethylene- and triethylene glycol) no positive increases in SCEs were observed following exposure to a similar or higher range of osmolalities (BRRC reports 44-56; 47-94; 49-83).

The observation of low level but statistically significant increases in SCEs in tests both with and without S9 activation indicated that tetraethylene glycol produced positive increases in SCEs following the criteria employed to evaluate this test at BRRC. However, the biological significance of these weakly positive effects must be interpreted with caution, both because of the lack of unequivocal dose-response data and the high osmolality conditions employed in the in vitro test system.