Reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-d

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames study is available for the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol.

Studies on TEG and TTEG, components of the reaction mass, and Crude Penta, a mixture similar to the reaction mass, are available.

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

HGPRT

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.

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.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

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 increase in SCEs, not dose responsive

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable study, meets basic scientific principles

Justification for type of information:

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

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 chromosome aberration test

Target gene:

CHO cells

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:

0.0, 35, 42, 50 mg/ml

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: cyclophosphamide and triethylene melamine

Details on test system and experimental conditions:

Dose selection: Appropriate concentrations for cytogenetic testing were determined by preliminary measurements of cytotoxicity to CHO cells using a broad range of concentrations from 1 - 50 mg/ml tested both the presence and absence of a rat liver S-9 metabolic activation system.
Selection of a suitable range of concentrations for testing was based upon an estimate of the doses which would not excessively inhibit mitotic cell invision of the treated cells. A maximum concentration of 50 mg/ml is tested for non-cytotoxic test chemicals.
Test procedure: For evaluation of direct clastogenic potential, CHO cells were exposed to TEG and appropriate controls for a continuous 6- or 10-hour period without S-9 activation. Indirect genotoxic potential, requiring metabolic activation by liver S-9 homogenate, was studied with a 2-hour exposure period to test chemical and S-9 activation system. Following the 2-h exposure period, cells were rinsed, fresh medium was added and cells were then harvested at 6 and 10 h after the start of exposure. Chromosomes were prepared by standard procedures. A total of 50 cells/culture/harvest interval was examined for chromosome damage using duplicate cultures for the test agent and solvent controls. At least 5 dose levels were tested both with and without metabolic activation. Incidence of chromosome damage was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division (mitosis).

Statistics:

Analyses of the test data employed the Fisher's Exact Test (one-tailed) to determine statistical significance and differences between the test and control populations.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results obtained in this study demonstrated that the test substance did not produce significant increases in the proportion of cells with chromosome aberrations. The predominant type of chromosome damage observed in this study was simple chromatid breakage. None of the other types of typical chromosome damage scored in this test system were remarkable different from normal variations generally encountered with these cultured cells.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative

Results obtained in tests with and without an S9 metabolic activation system indicated that the test substance did not produce 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 system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 28, 2016 - December 9, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strains).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fractions

Test concentrations with justification for top dose:

156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate
Top dose was selected because recommended top dose in the guidelines for testing in the Confirmatory Mutation Assay.vehicle

Vehicle / solvent:

Distilled water (Pusher/Pentaethylene glycol mixture and positive control material sodium azide). DMSO for positive controls 4-nitroquinoline 1-oxide, 0-aminoacridine hydrochloride hydrate, 2-nitrofluorene, and 2-aminoanthracene.

Untreated negative controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Initial Toxicity Mutation Assay
Before commencing the Confirmatory Mutation Assay, Pentaethylene Glycol Mixture was tested for cytotoxicity to all Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101). The experiment was conducted both in the absence and presence of metabolic activation (5% v/v S9 mix).
Tubes containing 2 mL of molten top agar 0.5 mM histidine/biotin were maintained at 45 ± 2 °C for Salmonella typhimurium tester strains. Tubes containing 2 mL of plain top agar were maintained at 45 ± 2 ºC for Escherichia coli WP2 uvrA (pKM101) tester strain and prior to the addition of the test item and controls, 100 µL of tryptophan solution (100 µg/mL) was added to the top agar tubes. Volumes of 100 µL of the relevant stock solution of test item and distilled water were used for treatment and negative control, respectively.
A volume of 500 µL of 5% v/v S9 mix was added in the presence of metabolic activation and 500 µL of 0.2 M phosphate buffer was added in the absence of metabolic activation. Finally 100 µL of bacterial culture was added to the tubes and mixed. Cultures used were checked for cell viability prior to testing. This treatment mixture was incubated in shaking water bath for
20 ± 2 minutes at 37 ± 1 °C. After incubation, 2 mL molten top agar was added to this treatment mixture and poured on MGA plates and allowed to solidify. Duplicate sets were maintained for each concentration of Pentaethylene Glycol Mixture, negative control and positive control. The petri plates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the background bacterial lawn pattern and reduction in number of colonies.

Confirmatory Mutation Assay
The Confirmatory Mutation Assay was conducted as an independent experiment. In the trial, the treatment was performed both in the absence and presence of metabolic activation (10% v/v S9 mix). The treatment was performed by the preincubation technique as described in the Initial Toxicity Mutation Assay. Triplicate sets of plates were treated for each test concentration of Pentaethylene Glycol Mixture, negative and positive controls.
The tester strains were exposed at the concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 µg Pentaethylene Glycol Mixture/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation for Confirmatory Mutation Assay, respectively.
Volumes of 100 µL of relevant stock solutions A – F were used to obtain the required test concentrations for treatment both in the absence and presence (10% v/v S9 mix) of the metabolic activation system.
Samples were collected and sent for analysis.
The numbers of revertant colonies were recorded approximately after the 48 h incubation period.
Treatment with 2-aminoanthracene in the absence of metabolic activation was performed for tester strain TA100 in both the trials to demonstrate the efficiency of the S9 fraction used in the study.

Analytical Verification and Stability of Test Item
Dose formulations of Pentaethylene Glycol Mixture were prepared in distilled water by the study director during the study. Dose formulations for the Initial Toxicity Mutation Assay and the Confirmatory Mutation Assay were not corrected for purity. Samples of dose formulations and the vehicle (distilled water) were sent to the Department of Chemistry, JRF for active ingredient concentration analysis for Confirmatory Mutation Assay.

Sampling
Samples were collected from all six prepared dose formulations along with vehicle (distilled water) for active ingredient concentration and lowest and highest dose formulation for stability analysis during the confirmatory mutation assay following the detailed procedures below:
Two sets of single replicates of 3 mL each of concentration (1562.5, 3125, 6250, 12500, 25000, and 50000 µg/mL) and vehicle (distilled water) were taken from middle portion for active ingredient concentration analysis at 0 hour. Two sets of single replicates of 3 mL each of highest concentration (50000 µg/mL) and lowest concentration (1562.5 µg/mL) were taken from middle portion for stability analysis at 4 hour of dose formulation preparation i.e. at the end of plate exposure. All the aliquots were stabilized by immediate freezing (-70 ± 10 °C) to quench any degradation of test item. The first sets of replicates were sent to Department of Chemistry (JRF) for analysis. Second set of replicates were stored in deep freezer (-70 ± 10 ºC) as backup. Due to extraction problem, back up samples were reanalysed using same analytical parameter.

Stability
Stability of the test item in distilled water was tested concurrently by test samples before the initiation of exposure and after completion of plate exposure (4 hours) by using the validated analytical method provided by the sponsor. All prepared concentrations were sampled before the initiation of plate exposure, while only the lowest and the highest test concentrations were sampled at 4 hours post dose formulation preparation for stability analysis.

Analysis
The samples collected during the confirmatory mutation study as described above were analysed for active ingredient concentration and stability.

Evaluation criteria:

Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535, and TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strains TA100 and Escherichia coli WP2 uvrA (pKM101)
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0- times the mean negative control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non mutagenic.

Species / strain:

other: S.typhimuriam TA1537, TA1535, TA98, TA100, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative Controls
The results of the study indicate that the values for the negative controls (Distilled Water) in all strains were within limits of historical range.

Positive Controls
2-Aminoanthracene was used as the positive control in the presence of a metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation.
Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges.This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (Initial Toxicity Mutation Assay and Confirmatory Mutation Assay) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity Mutation Assay
Bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate (two plates/concentration). Normal growth was observed up to the tested concentration of 5000 µg/plate both in the absence and presence of metabolic activation system (5% v/v S9 mix). No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control. Hence, 5000 µg Pentaethylene Glycol Mixture/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation system (10% v/v S9 mix), respectively, for Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101) tester strain.

Confirmatory Mutation Assay
From the results of the Initial Toxicity Mutation Assay, 5000 µg Pentaethylene Glycol Mixture/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation system (10% v/v S9 mix), for Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101) tester strain.
In the confirmatory mutation assay, bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate (three plates/concentration).
Normal growth was observed up to the tested concentration of 5000 µg/plate in the absence and presence of the metabolic activation system (10% v/v S9 mix) in all the Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101). No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Dose Formulation Analysis
Pentaethylene Glycol Mixture was found to be within acceptable range of ± 15% of nominal (% RSD < 10%) in distilled water at all the tested concentrations during confirmatory mutation assay. The doses complied with the presence of test item for its claimed concentration (± 15% of nominal) of active ingredient. Average recoveries were 97.2%, 98.7%, 95.5%, 103%, 104%, and 103% at test concentrations of 1562.5, 3125, 6250, 12500, 25000, and 50000 µg/mL, respectively. Results of stability analysis showed that the test item was stable at the end of treatment (4 hours) and recovery of the test concentrations 1562.5 and 50000 µg/mL (lowest and highest concentration) was 104% and 104% of the 0 hour concentration, respectively.

TABLE1:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Initial Toxicity Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate (Absence of Metabolic Activation)
	Salmonella typhimurium Tester Strains																			E.coli WP2 uvrA (pKM101)
	TA1537				TA1535					TA98					TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean		SD	Ratio	Lawn Pattern	Mean		SD	Ratio	Lawn Pattern	Mean		SD	Ratio	Lawn Pattern
NC(distilled water)	7.00	4.24	1.00	NI	19.00	7.07	1.00	NI	20.50		2.12	1.00	NI	123.00		16.97	1.00	NI	117.00		11.31	1.00	NI
1.5	5.00	1.41	0.71	NI	18.50	3.54	0.97	NI	18.50		3.54	0.90	NI	126.50		9.19	1.03	NI	110.00		2.83	0.94	NI
5	8.50	0.71	1.21	NI	20.00	2.83	1.05	NI	16.00		1.41	0.78	NI	115.00		8.49	0.93	NI	115.00		7.07	0.98	NI
15	8.00	1.41	1.14	NI	16.50	6.36	0.87	NI	19.50		3.54	0.95	NI	121.00		7.07	0.98	NI	111.00		4.24	0.95	NI
50	7.50	3.54	1.07	NI	18.00	2.83	0.95	NI	20.50		7.78	1.00	NI	121.50		10.61	0.99	NI	119.50		3.54	1.02	NI
150	7.00	2.83	1.00	NI	19.00	5.66	1.00	NI	18.00		2.83	0.88	NI	121.00		4.24	0.98	NI	117.50		7.78	1.00	NI
500	8.50	3.54	1.21	NI	18.00	2.83	0.95	NI	19.00		1.41	0.93	NI	128.00		4.24	1.04	NI	118.50		0.71	1.01	NI
1500	8.50	0.71	1.21	NI	17.50	0.71	0.92	NI	20.00		1.41	0.98	NI	119.50		6.36	0.97	NI	109.50		3.54	0.94	NI
5000	6.50	0.71	0.93	NI	16.00	5.66	0.84	NI	17.50		2.12	0.85	NI	126.50		2.12	1.03	NI	118.00		4.24	1.01	NI
PC	301.50	26.16	43.07	NI	322.50	33.23	16.97	NI	582.50		58.69	28.41	NI	893.50		21.92	7.26	NI	1092.50		24.75	9.34	NI
2-Aa	NA	NA	NA	NA	NA	NA	NA	NA	NA		NA	NA	NA	133.50		6.36	1.09	NI	NA		NA	NA	NA

Key:        SD = Standard deviation, NC = Negative control, PC = Positive control {for TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), for TA1535 = Sodium azide (0.5 µg/plate), for TA98 = 2-Nitrofluorene (7.5 µg/plate), for TA100 = Sodium azide (5 µg/plate), forE. coli WP2 uvrA (pKM101) =4-Nitroquinoline 1-oxide(0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),Test item = Pentaethylene Glycol Mixture, NI = No Inhibition

TABLE2:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Initial Toxicity Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	Salmonella typhimurium Tester Strains															E.coli WP2 uvrA (pKM101)
	TA1537				TA1535				TA98				TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern
NC (Distilled water)	9.00	4.24	1.00	NI	20.50	6.36	1.00	NI	29.00	9.90	1.00	NI	129.00	12.73	1.00	NI	118.50	9.19	1.00	NI
1.5	6.50	2.12	0.72	NI	16.50	2.12	0.80	NI	25.50	10.61	0.88	NI	123.50	6.36	0.96	NI	119.00	7.07	1.00	NI
5	5.00	2.83	0.56	NI	21.00	7.07	1.02	NI	25.50	4.95	0.88	NI	125.50	6.36	0.97	NI	116.00	7.07	0.98	NI
15	6.00	2.83	0.67	NI	19.50	2.12	0.95	NI	25.50	6.36	0.88	NI	125.50	7.78	0.97	NI	114.50	6.36	0.97	NI
50	8.50	2.12	0.94	NI	21.00	1.41	1.02	NI	21.50	2.12	0.74	NI	123.50	13.44	0.96	NI	116.00	4.24	0.98	NI
150	8.50	0.71	0.94	NI	20.50	3.54	1.00	NI	25.50	3.54	0.88	NI	125.00	4.24	0.97	NI	120.50	6.36	1.02	NI
500	4.50	0.71	0.50	NI	18.50	4.95	0.90	NI	25.50	4.95	0.88	NI	129.50	13.44	1.00	NI	118.00	5.66	1.00	NI
1500	9.50	2.12	1.06	NI	19.00	2.83	0.93	NI	23.00	7.07	0.79	NI	127.50	3.54	0.99	NI	119.50	4.95	1.01	NI
5000	5.50	0.71	0.61	NI	16.00	1.41	0.78	NI	21.50	2.12	0.74	NI	125.50	3.54	0.97	NI	117.50	3.54	0.99	NI
PC(2-Aa)	281.50	43.13	31.28	NI	354.50	47.38	17.29	NI	564.50	60.10	19.47	NI	981.00	48.08	7.60	NI	1161.50	178.90	9.80	NI

Key:    SD = Standard deviation, NC = Negative control, PC = Positive control, 2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535,E. coli WP2 uvrA(pKM101) and 5 µg/plate for TA98 and TA100),Test item = Pentaethylene Glycol Mixture, NI = No Inhibition

TABLE3:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate (Absence of Metabolic Activation)
	Salmonella typhimurium Tester Strains																E.coli WP2 uvrA (pKM101)
	TA1537				TA1535				TA98				TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern
NC (Distilled water)	8.00	4.00	1.00	NI	17.00	5.29	1.00	NI	21.67	7.37	1.00	NI	127.00	13.53	1.00	NI	124.67	9.07	1.00	NI
156.25	7.33	3.51	0.92	NI	16.00	5.29	0.94	NI	20.67	7.64	0.95	NI	129.33	16.56	1.02	NI	123.33	8.50	0.99	NI
312.5	8.67	3.51	1.08	NI	16.00	4.58	0.94	NI	22.67	7.37	1.05	NI	127.67	10.07	1.01	NI	126.67	7.09	1.02	NI
625	7.67	1.53	0.96	NI	17.67	6.03	1.04	NI	21.00	8.72	0.97	NI	131.00	10.15	1.03	NI	122.00	7.55	0.98	NI
1250	8.67	1.53	1.08	NI	16.33	4.16	0.96	NI	22.67	4.16	1.05	NI	125.00	7.00	0.98	NI	127.33	8.62	1.02	NI
2500	7.67	3.51	0.96	NI	18.00	5.29	1.06	NI	22.00	6.24	1.02	NI	133.33	9.50	1.05	NI	122.67	5.86	0.98	NI
5000	7.33	2.52	0.92	NI	16.67	4.73	0.98	NI	21.00	5.29	0.97	NI	126.33	11.06	0.99	NI	121.33	12.06	0.97	NI
PC	419.33	38.03	52.42	NI	542.33	43.32	31.90	NI	765.67	55.05	35.33	NI	1073.67	14.19	8.45	NI	870.00	115.01	6.98	NI
2-Aa	-	-	-	-	-	-	-	-	-	-	-	-	128.67	6.66	1.01	NI	-	-	-	-

Key:    SD = Standard deviation, NC = Negative control, PC = Positive control {for TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), for TA1535 = Sodium azide (0.5 µg/plate), for TA98 = 2-Nitrofluorene (7.5 µg/plate), for TA100 = Sodium azide (5 µg/plate), for E. Coli WP2 uvrA (pKM101) =4-Nitroquinoline 1-oxide(0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),Test item = Pentaethylene Glycol Mixture, - = Not applicable, NI = No Inhibition

TABLE4:Mean Count of His⁺and Trp⁺Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Confirmatory Mutation Assay)

Concentration of Test Item (µg/plate)	Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	Salmonella typhimurium Tester Strains																E.coli WP2 uvrA (pKM101)
	TA1537				TA1535				TA98				TA100
	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern	Mean	SD	Ratio	Lawn Pattern
NC (Distilled water)	9.33	4.51	1.00	NI	20.00	7.00	1.00	NI	23.33	6.81	1.00	NI	131.33	14.05	1.00	NI	131.00	10.82	1.00	NI
156.25	9.00	3.61	0.96	NI	20.33	4.04	1.02	NI	23.67	5.69	1.01	NI	134.00	9.17	1.02	NI	128.00	12.53	0.98	NI
312.5	9.33	3.21	1.00	NI	19.00	6.24	0.95	NI	22.67	6.51	0.97	NI	126.67	11.59	0.96	NI	132.33	9.02	1.01	NI
625	10.00	3.61	1.07	NI	19.33	5.13	0.97	NI	23.67	5.51	1.01	NI	137.00	10.82	1.04	NI	125.33	12.86	0.96	NI
1250	9.00	3.00	0.96	NI	20.67	5.69	1.03	NI	23.00	5.20	0.99	NI	125.00	10.82	0.95	NI	130.33	8.50	0.99	NI
2500	9.00	4.36	0.96	NI	20.33	6.81	1.02	NI	24.33	7.09	1.04	NI	133.00	11.14	1.01	NI	131.00	9.85	1.00	NI
5000	9.67	3.51	1.04	NI	19.00	7.21	0.95	NI	22.67	5.69	0.97	NI	130.00	14.53	0.99	NI	127.33	11.02	0.97	NI
PC	429.33	33.13	46.02	NI	629.00	56.56	31.45	NI	1005.67	61.91	43.11	NI	1175.00	87.61	8.95	NI	962.33	136.14	7.35	NI

Key:        SD = Standard deviation, NC = Negative control,PC = Positive control, 2Aa = 2-Aminoanthracene(10 µg/plate for TA1537, TA1535, E. coli WP2 uvrA (pKM101)and 5 µg/plate for TA98 and TA100), Test item = Pentaethylene Glycol Mixture, NI = No Inhibition

Conclusions:

From the results of this study, under the specified experimental conditions, Pentaethylene Glycol Mixture was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of Pentaethylene Glycol Mixture to induce reverse mutations in Salmonella typhimurium (strains: TA1537, TA1535, TA98, and TA100) and a tryptophan deficient strain, Escherichia coli WP2 uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method. 

Pentaethylene Glycol Mixture was tested in the absence and presence of metabolic activation using distilled water (DW) as the solvent. In the Initial Toxicity-Mutation Assay, bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration). No cytotoxicity was observed up to the tested concentration of 5000 µg/plate, with and without metabolic activation, in any of the five strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

From the results of the Initial Toxicity-Mutation Assay, 5000 µg/plate was selected, being the recommended top dose indicated in the guidelines for testing in the Confirmatory Mutation Assay. In the Confirmatory Mutation Assay, bacterial cultures were exposed to Pentaethylene Glycol Mixture at concentrations of 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate (three plates/concentration). No cytotoxicity was observed at all concentrations tested, with and without metabolic activation, in any of the five tester strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

Pentaethylene Glycol Mixture was found to be within acceptable range of ± 15% of nominal concentrations at the tested stock concentrations of 1562.5, 3125, 6250, 12500, 25000, and 50000 µg/mL during the Confirmatory Mutation Assay. Recoveries ranged between 95.5% to 104% at the above tested concentrations. Results of stability analysis showed that the test item was stable at the end of treatment (4 hours) and recovery of the test concentrations at 1562.5 and 50000 µg/mL (lowest and highest concentration) was 104% and 104% of the 0 hour concentrations, respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, Pentaethylene Glycol Mixture is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Studies on TTEG, a component of the reaction mass, and Crude Penta, a mixture similar to the reaction mass, are available.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

8 - 11 December 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to GLP and sufficient data is available for the interpretation of results.

Justification for type of information:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Seventy male and female, 11-12 week old Sprague-Dawley rats were obtained from Harlan Sprague Dawley Inc., Indianapolis, IN. This strain was used because of this laboratory's toxicological experience with Sprague-Dawley rats. For definitive testing, the male rats weighed between 290 g to 326 g and the female rats weighed between 187 g and 220 g at the initiation of the study. The animals were inspected for health upon arrival and were weighed prior to dosing. Only animals which appeared healthy were used for testing following a minimum of a 5-day acclimation period after arrival at the
laboratory. Routine quality control testing was performed on the animals under the direction of the BRRC clinical veterinarian and the animals were found to be healthy. Animals not used for these studies were sacrificed and discarded.

One to five rats/sex/cage were housed in undivided stainless steel Maxi-rack cages with wire-mesh floors under which animal cage board was placed. Prior to testing, all animals were assigned unique animal numbers, weighed and identified with monel ear tags. The animals were randomized and assigned to dosage groups using a stratified design to assure homogeneity of body weights between groups. After randomization, the rats were transferred to clean divided Maxi-tack cages and housed individually in the divided cages until sacrifice. The animals were fed commercial pelleted rodent diet (Agway BMH3000 Certified Rodent Pellets). Food was provided ad libitum until approximately 3:00 pm of the afternoon prior to dosing. Food was replaced 1-3 hr after dosing and provided ad Jibitum until sacrifice. Water was supplied by the Municipal Authority of Westmoreland County (Greensburg, PA) and available ad libitum. The animal room temperature and humidity was controlled and room lights were automatically timed for a 12-hr light/dark cycle. Room temperature and relative humidity were monitored continuously
throughout the the study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Control Agents:
The sponsor indicated that the test sample was infinitely soluble in water; thus, water was used as the solvent for dilution of the test agent. The volume of the vehicle control substance (water) was administered at an equivalent volume as that used to deliver the test substance (10 ml/kg). Cyclophosphamide (CAS# 6055-19-2) was administered as a single intraperitoneal injection of 30 mg/kg to demonstrate the responsiveness of the animals to a recognized clastogenic agent. Two groups of animals were treated with either the vehicle or the positive control agent. These groups were sacrificed and bone marrow was harvested only at the 24 hour interval after dosing.

Sample Preparation:
Stock dilutions were prepared fresh on the morning of each test day and the accuracy of dilution in definitive tests was verified by gravimetric analysis. Dilutions were prepared to achieve concentrations that would deliver a dosing volume of 10 ml/kg. The sponsor indicated that the sample was stable for at least one year in water and no additional stability or content analyses was performed in the solvent at BRRC.

Duration of treatment / exposure:

Rats were sacrificed 24 hours after dosing with vehicle or positive control agents and 12, 24 or 48 hours after dosing with tetraethylene glycol

Frequency of treatment:

A single dose was administered

Post exposure period:

Rats were sacrificed 24 hours after dosing with vehicle or positive control agents and 12, 24 or 48 hours after dosing with tetraethylene glycol

Remarks:

Doses / Concentrations:
1250, 2500 or 5000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females/dose level

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CAS# 6055-19-2) was administered as a single intraperitoneal injection of 30 mg/kg to demonstrate the responsiveness of the animals to a recognized clastogenic agent.

Tissues and cell types examined:

Bone marrow was examined.

Microscopic evaluation - When possible, 500 cells per animal were scored for proportion of mitotic cells. Reduced numbers of mitotic cells is an indication of cytotoxicity and excessive reduction in number of mitotic cells can preclude cytogenetic evaluation of chromosomes. Fifty metaphase cells were evaluated for incidence and type of chromosome damage for each animal/dose/sample time. Each cell was evaluated for chromosome number, specific type of chromosome- or chromatid-type aberrations and further classified for deletions and exchanges. Gaps, endoreduplicated
chromosomes and polyploid cells were noted and tabulated but are not included as aberrations when computing the proportion of aberrant cells or for use in statistical analyses. Severely damaged cells (>/= 10 breakage events) and pulverized cells were recorded as severely damaged (SD) but no attempt was made to classify the types of damage in these severely damaged cells.

Details of tissue and slide preparation:

Sacrifice and Bone Marrow Extraction
The animals were sacrificed at preassigned time intervals of 12 hr, 24 hr or 48 hr after administration of the dosing material. Colchicine (4 mg/kg) was injected IP 2-3 hours prior to sacrifice. Animals were euthanized by Metofane inhalation and/or cervical dislocation. A femur was removed from each animal and the bone marrow was flushed into a centrifuge tube using 10-15 ml of freshly prepared Hank's Balanced Salt Solution (pH 7.0). The suspension was centrifuged, then the pellet was resuspended in 15 ml of 0.075M KCl (hypotonic) solution and incubated at 37OC for 20-30 minutes. Cells were centrifuged and fixed with 2-3 changes of Carnoy's fixative (3:l methanol; acetic acid). Fixed cells were refrigerated at 4OC at least 12 hours prior to slide preparation. Just prior to preparing slides, cells were centrifuged and resuspended in approximately 1 ml of Carnoy's fixative to achieve an opalescent suspension. To prepare slides, 3-4 drops of the suspension were dropped onto a clean wet slide and air-dried. Slides were identified by BRRC chemical number, animal number and sacrifice interval. Initially, one slide was prepared for each animal. Additional slides were prepared as needed to assure that sufficient numbers of mitotic cells were available. Chromosomes were stained for approximately 10 minutes in a dilute Giemsa solution (1:25). Slides were rinsed with water, dried and coverslipped.

Evaluation criteria:

A positive effect was considered to be a statistically significant and
dose-related increase in the frequency of structural chromosome aberrations.
Alternatively, the production of a statistically significant increase for at least one dose level was considered to be an equivocal effect if there was no evidence of a dose-related response. A test substance which did not produce positive increases as described above was considered to be inactive as an in vivo clastogenic agent in this test system.

Statistics:

Analyses of the test data employed the Fisher's Exact Test (one-tailed) to determine statistical significance of increased incidences of aberrant cells 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 cytogenetic data often vary from a normal distribution required for parametric analyses. The total number of aberrant cells from all animals at each concentration were compared to the respective solvent control value for each sex at each sample time. A difference between treated and control cells was considered to be significant when p p>0.01; b = 0.01 > p > 0.001; c = p < 0.001.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Remarks:

Animals were tested at 5000 mg/kg, the highest dose this laboratory will examine.

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Selection of Dose Levels:
In a limited toxicity test, male and female Sprague-Dawley rats were administered a single peroral dose of 5000 mg/kg of tetraethylene glycol and observed for clinical signs. No mortality or adverse clinical signs were observed, In addition, body weights were obtained at the end of the 5 day observation period and all animals showed weight gains of at least 16 g. Since tetraethylene glycol was demonstrated to be relatively non-toxic, the maximum limit dose (5000 mg/kg) was selected as the highest dose level for definitive testing. Subsequent dose levels of 2500 mg/kg and 1250 mg/kg were selected at intervals of 50% and 25% of the maximum limit dose, respectively.

Evaluation of Potential Chromosome Damage:
Table 1 presents the incidence of chromatid-type and chromosome-type aberrations observed in bone marrow cells of male and female rats obtained 12 hr, 24 hr and 48 hr after dosing, respectively. Relatively low frequencies of simple chromatid breaks and chromosome fragments were the predominant types of damage observed at all three sample periods with both male and female rats.

Table 2 presents a general summary of the overall test results and a statistical summary of the significance of the differences observed. No
statistically significant or dose-related increases in chromosome aberrations above vehicle control values were observed with either male or female rats sacrificed at any of the three time intervals after dosing. Mean incidence of aberrant cells ranged from 1.2% to 5.2% for male rats, and from 0.8% to 4.8% for female rats administered tetraethylene glycol. These incidences are within the typical range of background variability observed for this test system at BRRC .

Animals treated with the positive (cyclophosphamide) and vehicle (water) control agents were evaluated for chromosome damage at the 24 hr sample period. Cyclophosphamide was highly effective in producing significant numbers and types of damage with both male and female rats which demonstrates the responsiveness of the test animals to a known clastogenic agent. The incidence and types of aberrations found in bone marrow cells of rats dosed with the vehicle control were within the expected range of values for this test system consistent with a valid test.

Table 1 Summary of Chromosome Damage: In Vivo Cytogenetic Testing with Bone Marrow Cells of Rats

Test Material	Mitotic Index (%)	% Aberrant* Cells	Mitotic Index (%)	% Aberrant* Cells
Tetraethylene glycol, mg/kg	Males		Females
	12 hour values
1250	4.4 (1.0)	4.4 (2.97)	5.0 (0.8)	2.8 (2.68)
2500	3.7 (1.4)	2.4 (2.61)	4.3 (2.0)	0.8 (1.10)
5000	4.5 (1.5)	2.0 (1.41)	4.6 (1.7)	2.8 (2.28)
	24 hour values
1250	5.3 (0.6)	3.6 (2.19)	3.2 (1.4)	4.8 (4.15)
2500	4.9 (1.0)	3.6 (3.29)	5.9 (1.4)	3.6 (1.67)
5000	4.6 (1.2)	3.6 (3.58)	4.9 (0.5)	1.6 (1.67)
Control (Water)	5.1 (0.3)	3.6 (3.85)	5.4 (1.7)	3.2 (2.68)
Positive Control (Cyclophasphamide)	2.4 (0.5)	39.2 (4.15)	2.1 (1.2)	36.4 (4.34)
	48 hour value
1250	4.9 (1.6)	5.2 (3.03)	5.0 (1.5)	2.4 (2.61)
2500	5.2 (1.0)	1.2 (1.79)	6.5 (2.2)	1.2 (1.79)
5000	5.0 (1.3)	2.0 (2.00)	5.9 (0.7)	2.8 (2.28)

*A total of 50 cells/animal were evaluated.

Table 2 Tetraethylene Glycol: Summary of Chromosome Aberration Data and Statistical Analyses

Concentration		Number of	Total Number of	Total Number of	Mean %
mg/kg	Sex	Animals	Cells Scored	Aberrant Cells	Aberrant Cells (+S.D.)*
Tetraethylene glycol	12 Hr Sample Period
1250	M	5	250	22	4.4 (2.97)
	F	5	250	14	2.8 (2.68)
2500	M	5	250	12	2.4 (2.61)
	F	5	250	4	0.8 (1.10)
5000	M	5	250	10	2.0 (1.41)
	F	5	250	14	2.8 (2.28)
	24 Hr Sample Period
1250	M	5	250	18	3.6 (2.19)
	F	5	250	24	4.8 (4.15)
2500	M	5	250	18	3.6 (3.29)
	F	5	250	18	3.6 (1.67)
5000	M	5	250	18	3.6 (3.58)
	F	5	250	8	1.6 (1.67)
Control (Water)	M	5	250	18	3.6 (3.85)
	F	5	250	16	3.2 (2.68)
Positive Control (Cyclophosphamide)	M	5	250	196	39.2 (4.15)^c
	F	5	250	182	36.4 (4.34)^c
	48 Hr Sample Period
1250	M	5	250	26	5.2 (3.03)
	F	5	250	12	2.4 (2.61)
2500	M	5	250	6	1.2 (1.79)
	F	5	250	6	1.2 (1.79)
5000	M	5	250	10	2.0 (2.00)
	F	5	250	14	2.8 (2.28)

* Statistical significance above concurrent control values were analysed using the Fisher's Exact Test (1-tailed). c = p < 0.001.

Conclusions:

Interpretation of results (migrated information): negative
Tetraethylene glycol did not produce evidence of statistically significant or dose related increases in incidences of bone marrow cells with chromosome aberrarions in either male or female Sprague Dawley rats. Thus, tetraethylene glycol was not considered to be clastogenic under the conditions of he study.

Executive summary:

Tetraethylene glycol was administered to both male and female Sprague-Dawley rats by a single oral gavage and bone marrow cells were evaluated for potential chromosome damage. Test concentrations were chosen on the basis of a preliminary toxicity test which indicated that tetraethylene glycol was relatively nontoxic up to the maximum concentration of 5000 mg/kg which has been established as the BRRC limit dose for testing relatively nontoxic chemicals in this test system. Thus, three dose levels were tested in the definitive chromosome aberration test at intervals of 100%, 50% and 25% of the maximum dose of 5000 mg/kg.

None of the three dose levels of tetraethylene glycol tested produced statistically significant or dose related increases in relative numbers of chromosome aberrations compared to control values with either male or female Sprague-Dawley rats. Simple chromatid breaks and fragments were the predominant types of aberrations observed and the frequencies of these aberrations were within the range of the spontaneous incidence for this test system at this laboratory. Thus, tetraethylene glycol was not considered to be

clastogenic (chromosome breaking) to Sprague-Dawley rats under the conditions of this vivo test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro

In a key study, the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol was tested in a bacterial Ames test. S. typhimurium strains TA1537, TA1535, TA98, and TA100 and E. coli WP2 uvrA (pKM101) were used to test the mixture at concentrations of 156. 25, 312.5, 625, 1250, 2500, and 5000 µg/plate, with and without metabolic activation. No mutagenicity or cytotoxicity were observed in any tester strain up to the limit concentration.

In supporting studies, triethylene glycol (TEG) and tetraethylene glycol (TTEG), components of the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol, have been tested in Ames assays, as has a similar mixture of ethylene glycols, Crude Penta, comprised of 26% TTEG and 26.2% pentaethylene glycol. S. typhimurium strains TA98, TA100, TA1535, and TA1537 were used to test TEG for mutagenic activity at concentrations of 1.0-112.6 mg/plate with and without S9 in an Ames assay by Bushy Run Research Center (UCC, 1986a). No mutagenic activity was observed. An Ames tests performed with TEG (BASF, 2012) tested at up to 5 mg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without metabolic activation, similarly showed negative results. Mutagenicity of TTEG was tested in S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 at concentrations of 1.0-112.6 mg/plate with or without metabolic activation using triplicate cultures at each dose level (Bushy Run Research Center, 1986b). Mutagenicity of Crude Penta was tested in S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, with and without metabolic activation, at concentrations ranging from 0.10 to 10 mg/plate, without evidence of mutagenic activity (Bushy Run Research Center, 1991).

In additional key studies, TEG and TTEG have been tested in in vitro mammalian chromosome aberration tests. TEG was negative for genotoxic and cytotoxic effects in a mammalian chromosome aberration assay in CHO cells wherein concentrations of 35-50 mg/ml TEG were tested with and without metabolic activation (Bushy Run Research Center, 1986c). TTEG was also negative in the mammalian CHO/HGPRT forward mutation assay at concentrations up to 50 mg/ml (Bushy Run Research Center, 1987).

In a supporting study, a sister chromatic exchange (SCE) assay with CHO cells showed negative results at concentrations of up to 50 mg/ml TEG (Bushy Run Research Center, 1986d), with and without metabolic activation.

In a supporting study by Bushy Run Research Center (1987b), a weak response in the chromosome aberration test was seen in cultured CHO cells at concentrations of 24-50 mg/ml, with and without metabolic activation. However, the lowest dose tested in this study was 5x higher than the limit dose recommended by the current guidelines. Moreover, the high osmolality observed in this study (423 to 605 mOsm/kg H₂O) is a known confounder in this in vitro cell system. Therefore, this study is not considered appropriate for evaluation of classification of TTEG. TTEG produced chromosome aberrations in Chinese hamster epithelial liver (CHEL) cell line, which reportedly retains sufficient metabolic activation capability, but is not a typical cell line used for genotoxic study battery (Biondi et al., 2002). Positive results were also reported in CHO cells in the presence of metabolic activation in this study, but this result does not agree with the Union Carbide Corporation 1987 Klimisch 1 study. Finally, TTEG produced a weak but statistically significant increase in the incidence of SCEs in CHO cells at 1 to 50 mg/ml with and without metabolic activation; however, there was no dose-response associated with the incidence of SCEs (Bushy Run Research Center, 1987a). Again, the biological significance of these results should be evaluated with caution due to the high range of osmotic concentrations evaluated in the test system.

In Vivo

In the key study, TTEG was assessed in an in vivo bone marrow chromosome aberration assay in which male and female Sprague-Dawley rats were given doses up to 5000 mg/kg TTEG by gavage (Bushy Run Research Center, 1988). Dose levels of 1250, 2500, and 5000 mg/kg TTEG, chosen on the basis of a preliminary test which indicated that TTEG was relatively nontoxic up to the maximum concentration of 5000 mg/kg, produced no statistically significant or dose related increases in relative number of chromosome aberrations compared to control values with either male or female Sprague Dawley rats. Simple chromatid breaks and fragments were the predominant types of aberrations observed and the frequencies of these aberrations were within the range of the spontaneous incidence for this test system at this laboratory. Thus, TTEG was not considered to be clastogenic to Sprague-Dawley rats under the conditions of this in vivo test system.

 In a supporting study, an in vivo micronucleus (MN) test was performed in male and female Swiss-Webster mice given doses of 2500, 4000, or 5000 mg/kg TTEG i.p. (Bushy Run Research Center, 1987). No statistically significant difference in micronucleated polychromatic erythrocyte (MNPCE) frequency was observed in the TTEG treated mice at any time point, when compared to the concurrent vehicle controls.

 TTEG was also negative in a dominant lethal test by Bushy Run Research Center (1993). In this supporting study, male rats were treated for five days with 5000, 25000 or 50000 ppm TTEG in the drinking water and subsequently mated to naïve (untreated) females over ten weeks, with females replaced weekly. TTEG produced evidence of toxicity in males at 50000 ppm and urinary findings consistent with an osmotic diuresis at 25000 and 50000 ppm. However, no indications of subsequent reproductive or gestational effects were observed, including no significant pre-implantation loss over the ten week period. The NOEL for general toxicity was 25000 ppm.

In a supporting in vivo MN study in male and female Swiss Webster mice, i.p. injection of 2500, 4000, or 5000 mg Crude Penta/kg bw produced no increase in micronucleated polychromatic erythrocytes (Bushy Run Research Center, 1992).

Justification for classification or non-classification

The reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol was not mutagenic in an in vitro bacterial system. TEG and TTEG, components of the reaction mass, and similar mixture Crude Penta, were also not mutagenic in in vitro bacterial test systems. TEG were negative in mammalian chromosome aberration and sister chromatic exchange assays. TTEG displayed evidence of clastogenic effects in vitro, but this was with high doses (many times the current guideline maximum dose) confounded by high osmolality which are known clastogenicity inducers. Similarly, increased incidence of SCE after TTEG exposure occurred under conditions of high osmolality. In vivo MN studies with TTEG, however, produced no clastogenic effects in male or female Swiss-Webster mice or Sprague-Dawley rats and TTEG was negative in the dominant lethal test. Similarly, ethylene glycol mixture Crude Penta also did not produce increased MN in vivo. Based on these component data, genetic toxicity is not expected for the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol.