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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March - 20 March 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: Environmental Protection Agency, Health Effects Test Guidelines, HG-Gene Muta-S. QLBimiAm, EPA Report No. 560/6-84-002, October, 1984.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation 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-[2-(2-hydroxyethoxy)ethoxy]ethoxy}ethan-1-ol

Test material form:

other: Slightly viscous, water clear liquid

Details on test material:

Tetraethylene glycol was obtained from Union Carbide Corporation. Gas chromatographic analysis performed by the sponsor indicated that the sample was approximately 99.69% pure and contained 0.056% triethylene glycol and 0.058% pentaethylene glycol.

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats, was purchased from Microbiological Associates8 Bethesda, MD.

Test concentrations with justification for top dose:

0, 1, 3, 10, 30 and 112.6 mg/plate

Vehicle / solvent:

water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without S9 used 4-nitro-o-phenylenediamine for TA98 and TA1538, sodium azide for TA100 and TA1535, and 9-aminoacridine for TA1537. With S9 used 2-aminoanthracene (2-anthramine) for all strains.

Details on test system and experimental conditions:

Toxicity Testing
Sterile tubes are prepared containing 2 ml soft agar (6 g/1 agar and 5 g/1 NaCl) with a final concentration of 0.05 mM L-histidine and 0.05 mM D-biotin (the entire mixture is called top agar). Dilutions of the test chemical are made in an appropriate solvent so that the correct amount of chemical can be added to each tube in 100 ul amounts. If less than 100 ul amounts of the test chemical is used, phosphate-buffered saline is used to adjust the total volume to 100 ul.

At least five doses are tested with maximum doses of 50 mg (for solids) or 100 ul (for liquids) per plate for nontoxic chemicals, unless limited by solubility. Generally, a chemical cannot be considered to be nonmutagenic unless at least 5 mg/plate has been tested, unless limited by toxicity or solubility (deSerres and Shelby, 1979; HG-Gene Muta-S. tvphimuriumf October, 1984). A 100 ul aliquot of an overnight broth culture of strain TA100 is added followed by the appropriate amount of test chemical. The mixture is vortexed and poured onto the surface of a Vogel-Bonner Medium E agar plate (VB-E plate). The top agar is allowed to harden and the plates are incubated at 37°C for 24 to 48 hours. The plates are examined for the condition of their background lawns and growth is recorded as either confluent, sparse, or absent. Confluency is considered an indication of nontoxicity, sparse growth indicates moderate toxicity, and lack of growth is recorded as toxic.

Controls
Concurrent solvent and positive controls are run with each test. The solvent control is the maximum amount of solvent added with the diluted test chemical. For chemicals tested without dilution (i.e., by direct addition), water is used as the solvent control. Both activation-dependent and activation-Independent positive controls are used. The activation-independent controls are 4-nitro-o-phenylenediamine for TA98 and TA1538, sodium azide for TA100 and TA1535, and 9-aminoacridine for TA1537. The activation-dependent control in 2-aminoanthracene (2-anthramine) for all strains. The concentrations are determined from prior dose-response experiments on these chemicals and are listed in the tables of results in this report.

Sample Preparation:
An initial stock solution of the test substance was prepared by mixing tetraethylene glycol in water to achieve a concentration of 300 mg/ml. All subsequent dilutions were made in the same solvent. Dilutions of the test substance were made fresh each day of testing. All dilutions for the mutagenicity tests were analyzed gravimetricaHy to determine actual concentrations.

Testing:
The test chemical was tested in triplicate at five doses chosen to span a range which included moderately toxic to relatively nontoxic concentrations. The test substance was nontoxic in the preliminary toxicity test; thus, 100 ul of liquid was used as the maximum dose. Testing was performed both with and without metabolic activation. Concurrent solvent and positive controls were tested in each experiment.

Data Collection
Bacterial colonies are counted manually or by an Artek Model #880 Colony Counter, or equivalent. The counter is calibrated for each test to check the
counting accuracy. The numbers of colonies per plate are counted and recorded. An examination is also made of the background lawn on each plate. If toxicity is observed as an inhibition of growth of the background lawn, the plate is not counted, but is recorded as toxic. If the background lawn is sparse and the colony count is still recorded, that number is not used in the calculation of the mean number of colonies and its standard deviation. A reduction in the number of spontaneous revertant colonies is also an indication of toxicity, and these are labelled "toxic" when the average number of colonies is less than one half of the average number for the solvent control.

Evaluation criteria:

The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative. If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen,

Statistics:

Means and Standard Deviations were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Tetraethylene glycol was tested in a preliminary toxicity screen at 112.6, 30, 10, 5, 3, 1, 0.3, 0.1, 0.03, and 0.01 milligrams per plate with strain TA100 only. When possible, the highest dose level for definitive testing is selected to produce some degree of cytotoxicity based on the preliminary toxicity test results. However in this study, no cytotoxicity was evident in the preliminary test and tetraethylene glycol did not decrease either the number of revertant colonies or the growth of the background lawn. Thus, a testable dose of 100 ul/plate (112.6 mg/plate) was the highest dose level tested and four additional half-log doses ranging from 1.0 mg/plate to 30 mg/plate were tested in the definitive mutagenicity experiments.

Numbers of revertant colonies per plate and the respective means and standard deviations of each dose for each strain of bacteria are shown in Table 1 (without activation) and in Table 2 (with activation). No indication of mutagenicity was observed at any of the tested doses, either by evidence of a dose-response relationship or a doubling of the number of colonies over the solvent control.

All strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain, and the number of spontaneous revertants was within the historical range of variation observed at this laboratory (see Appendix 3). All positive and negative controls were tested concurrently with the test chemical. Concurrent sterility test results showed that the mixture of S9, PBS, the test chemical and all solvents and controls were sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY - Without Activation

Test Material	Dose per plate	Mean + S.D.
TA98
Solvent (water)	100 mg	27 + 6.4
4 -NPD	0.01 mg	1038 + 20.4
TTEG	1 mg	31 + 2.1
TTEG	3 mg	35 + 7.0
TTEG	10 mg	29 + 6.8
TTEG	30 mg	37 + 6.7
TTEG	112.6 mg	39 + 5.5
TA100
Solvent (water)	100 mg	125 + 6.4
NaN3	0.01 mg	1837 + 37.0
TTEG	1 mg	109 + 8.7
TTEG	3 mg	124 + 7.8
TTEG	10 mg	127 + 20.4
TTEG	30 mg	136 + 1.0
TTEG	112.6 mg	128 + 18.0
TA1535
Solvent (water)	100 mg	38 + 6.4
NaN3	0.01 mg	1800 + 24.0
TTEG	1 mg	39 + 2.0
TTEG	3 mg	50 + 1.5
TTEG	10 mg	40 + 4.7
TTEG	30 mg	40 + 6.7
TTEG	112.6 mg	37 + 8.1
TA1537
Solvent (water)	100 mg	5 + 1.5
9 -AA	0.06 mg	84 + 15.3
TTEG	1 mg	5 + 1.0
TTEG	3 mg	7 + 1.7
TTEG	10 mg	7 + 2.6
TTEG	30 mg	5 + 1.0
TTEG	112.6 mg	8 + 1.5
TA1538
Solvent (water)	100 mg	8 + 1.5
4 -NPD	0.01 mg	1001 + 86.9
TTEG	1 mg	8 + 1.0
TTEG	3 mg	6 + 0
TTEG	10 mg	6 + 1.5
TTEG	30 mg	8 + 4.0
TTEG	112.6 mg	9 + 4.2

TTEG: Tetraethylene glycol

4-NPD: 4-NITRO-O-PHENYLENEDIAMINE;

NaN₃: SODIUM AZIDE

9-AA: 9-AMINOACRIDINE

Table 2 RESULTS OF THE SALMONELLA MUTAGENICITY ASSAY - With Activation

Test Material	Dose per plate	Mean+S.D.
TA98
Solvent (water)	100 mg	26 + 4.0
2 -AA	0.01 mg	2284 + 195.8
TTEG	1 mg	28 + 2.5
TTEG	3 mg	33 + 2.6
TTEG	10 mg	25 + 2.3
TTEG	30 mg	33 + 6.0
TTEG	112.6 mg	29 + 7.0
TA100
Solvent (water)	100 mg	91 + 11.8
2 -AA	0.01 mg	1409 + 228.3
TTEG	1 mg	101 + 5.2
TTEG	3 mg	83 + 3.6
TTEG	10 mg	89 + 12.9
TTEG	30 mg	90 + 12.1
TTEG	112.6 mg	102 + 14.4
TA1535
Solvent (water)	100 mg	12 + 3.2
2 -AA	0.01 mg	120 + 19.3
TTEG	1 mg	11 + 4.9
TTEG	3 mg	11 + 3.2
TTEG	10 mg	12 + 5.5
TTEG	30 mg	10 + 2.3
TTEG	112.6 mg	11 + 2.5
TA1537
Solvent (water)	100 mg	7 + 1.5
2 -AA	0.01 mg	166 + 46.1
TTEG	1 mg	5 + 1.0
TTEG	3 mg	5 + 2.3
TTEG	10 mg	4 + 2.1
TTEG	30 mg	5 + 1.7
TTEG	112.6 mg	7 + 0.6
TA1538
Solvent (water)	100 mg	11 + 3.1
2 -AA	0.01 mg	1010 + 187.9
TTEG	1 mg	18 + 4.0
TTEG	3 mg	15 + 3.8
TTEG	10 mg	19 + 3.8
TTEG	30 mg	21 + 4.9
TTEG	112.6 mg	17 + 3.0

TTEG: Tetraethylene glycol

2-AA: 2-AMINOANTHRACENE

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Tetraethylene glycol did not produce a dose dependent mutagenic response in any of the Salmonella typhimurium strains that were tested with or without a metabolic activation system. Under the conditions of this assay, tetraethylene glycol was not mutagenic in the Salmonella/microsome mutagenicity assay.

Executive summary:

Tetraethylene glycol was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test doses for the Ames test were chosen from data obtained in a preliminary study which indicated that all concentrations tested including the maximum concentration established by BRRC standard protocol for this test system (100 μl/plate), allowed confluent growth of the background lawn and did not produce evidence of cytotoxicity. In the definitive mutagenicity testing five concentrations of tetraethylene glycol ranging from 1.0 mg/plate to 112.6 mg/plate were tested with or without metabolic activation using triplicate cultures at each dose level.

Mutagenic activity was not observed with any of the five bacterial strains tested with or without metabolic activation. Tetraethylene glycol was not considered to be mutagenic in this in vitro bacterial test system.