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

Genetic toxicity: in vivo

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

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

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: clear, viscous liquid

Details on test material:

Tetraethylene Glycol was obtained from Union Carbide Corporation, Texas City, Texas. Analytical information provided by the sponsor indicated that the sample was approximately 99.08% tetraethylene glycol and contained approximately 0.92% triethylene glycol.

Test animals

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.

Administration / exposure

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

Doses / concentrations

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.

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.