2,2'-(ethylenedioxy)diethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Triethylene glycol was tested for its mutagenic potential in Salmonella typhimurium and Escherichia coli, in the standard plate test (SPT) and the preincubation test (PIT), according to the OECD TG 471 (BASF, 2012). Under the experimental conditions chosen, the test item is not mutagenic in the bacterial reverse mutation assay in the absence and presence of metabolic activation.
Further in vitro data on triethylene glycol are available (BRRC, 1986). These data refer to a negative chromosome aberration assay and a negative sister chromatid exchange assay.
Further, the read-across substance Diethylene glycol was not mutagenic in an HGPRT assay with and without metabolic activation (UCC 1984).

All available in vitro data revealed that the test item is not a mutagenic/genotoxic substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Analyses supplied by the sponsor indicated that the sample contained (by weight) 99.3% diethylene glycol, 0.09% monoethylene glycol, 0.39% triethylene glycol, 0.03% water, and 0.19% unknown components. The specific gravity was 1.1184. For the definitive tests, the test chemical was added directly into the cell culture medium of the test systems. Diethylene glycol is infinitely soluble in water with no solvent incompatibility as indicated in the information supplied by the sponsor. No other analyses of stability or concentration in the test system were performed.

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

S9 liver homogenate, prepared from Aroclor l254-induced, Sprague-Dawley male rats, as purchased from Microbiological Associates (MBA), Bethesda, MD. The S9 preparation was screened for metabolic activity by the supplier and at BRRC prior to use in our general testing program. Data from MBA showed that the S9 preparation was active with three different activation dependent mutagens in Salmonella bacterial strains TA98 and TA100.

Test concentrations with justification for top dose:

<= 50 mg/mL

Vehicle / solvent:

Test chemical was added directly into the cell culture medium of the test systems.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine and ethylmethanesulfonate

Details on test system and experimental conditions:

Selection of Test Concentrations:
Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would not kill over 90% of the treated cells. Test results wíth diethylene glycol indicated that concentrations up to 50 mg/ml, tested respectively with and without S9 metabolic activation, produced no excessive cytotoxic effects upon cell culture growth or mitotic indices. The 50 mg/ml (5.°G w/v) dose is the maximum dose used in the standard BRRC protocol and it was the highest dose tested with and without S9 activation in this study.


Test Procedures (HPGRT):
At 2 to 3 day intervals after treatment with the test agent, approximately 3 to 5 x 10^5 cells are subcultured in 100 mm tissue culture dishes in F12-D5 medium and incubated at 37°C in a 5 to 6% C02 atmosphere . After a period of at least 7 days to allow "expression" the mutant phenotype, cells of are dissociated with 0.05% to 0.075% trypsin, counted and plated at a concentration of 2 x 10^5 cells/dish in five 100 mm culture dishes (1 x 10^6 total cells) which each contain 10 ml of F12-D5 (TG) selective medium. At this same time interval, cells are diluted and 100 cells/dish are added to four culture plates containing F12-D5 medium (without TG) to assess viability (plating efficiency) of the treated cell population; the value obtained for the viable fraction for cells exposed to various doses and to control agents is used to correct the mutant frequency for cell populations with variable growth ability. All cultures are then incubated for an additional 6 to 8 days to allow growth of cells; medium is then discarded and colonies are fixed and stained for counting. The number of colonies in selection plates and in the viability test are counted by electronic methods, and data are recorded both as mutants/10^6 total cells and mutants/10^6 viable cells

Test Procedures (SCE Assay):
For testing without without metabolic activation, between 2 to 3 x 10^5 cells are plated into 75 cm2 culture flasks in F12-5 medium approximately 40-48 hrs prior to treatment and then incubated at 37°C in a 5 to 6% C02 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and 3 µg/ml BrdU is added to all flasks. Cells are treated with test agents for 5 hrs, media is then removed by suction, cells are rinsed with buffered, physiological salt solution and fresh medium containing 3 µg/ml BrdU is added for at least 24 hrs of additional incubation at 37°C to allow two rounds of cell division. Cells are then harvested and chromosomes are prepared for SCE staining.
Treatment of cells for testing of chemicals which require metabolic activation for mutagenic effectiveness is performed similarly as for treatments without activation, except for three modifications :
1. Approximately 40 to 48 hrs prior to exposure to the test agents, between 2 to 3 x 10^5 cells are added to F12-5 medium in 75 cm2 culture flasks.
2. Before treatment with the test agents, F12-5 medium is removed and F12
medium without serum is added.
3. S9 metabolic activation mixture is added to each flask (including solvent and positive controls) before addition of test agents.
4. Cells are treated for a total of 2 hrs (rather than 5 hrs as in tests without S9) and then incubated for at least 28 additional hours before harvest for chromosome preparation. The exact harvest time can vary depending upon the cytotoxicity of the chemical.

Control Agents:
Positive and solvent control materials were tested concurrently with the test sample to assure the sensitivity of the test system. For the CHO and SCE assays, dimethylnítrosamíne (DMN)-CAS 62-75-9 and ethylmethanesulfonate
(EMS)-CAS 62-50-0 were used as the positive control agents to assure the reliability and sensitivity of the test system for detecting metabolic activation dependent and independent mutagens, respectively. Cell culture medium was used as the vehicle and solvent control agent for this test chemical.

Evaluation criteria:

HPGRT:
The criteria for interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response effect following treatment. When a definite dose-response relationship is not evident but one oi more marginally significant values are obtained, a careful examination of the data from the concurrent positive and negative controls and comparisons to historical control data are used to evaluate the possible biological significance of the responses. Historical negative control data indicate that an average spontaneous mutation frequency in CHO cells of approximately 3 to 4 mutants/10^6 viable cells with a 95 percentile range of 0 to 18 mutants/10^6 viable cells. Statistical comparisons against unusually high or low
spontaneous controls are subjectively scrutinized in respect to the above
expected variability of this test system. Tests are usually repeated to clarify
the biological significance of the data when questionable results are obtained.

For evaluation criteria SCE, see "Any other information on materials and methods incl. tables"

Statistics:

The data were analyzed after transformation of the mutation frequencies (MF) and SCE values according to the conversion method of Box and Cox (1964). 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. 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:

negative

Cytotoxicity / choice of top concentrations:

other: > 50 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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. The authors observed that diethylene glycol was not highly cytotoxic when tested either with or without S9 metabolic activation. A concentration of 50 mg/ml produced 16% inhibition of growth without S9 and 27% inhibition with S9. For the definitive tests, a concentration range between 30 to 50 mg/ml was tested in the mutagenicity tests with and without S9. The 50 mg/ml (5% w/v) dose is the usual maximum dose for non-cytotoxic chemicals tested by the BRRC Standard Protocol to avoid possible artefacts produced by non-physiological cell-culture conditions at higher doses.

Executive summary:

The test substance was evaluated for potential genotoxic activity using the Chinese Hamster Ovary (CHO) Mutation test and the Sister Chromatid Exchange (SCE) test. The results indicated that the test substance did not produce a dose-related or repeatable, significant mutagenic effect in either of the in vitro screening tests employed. No detectable cytotoxicity to CHO cells was produced by the test substance even at the maximum concentration of 50 mg/mL (5 % w/v) typically evaluated as the highest dose in these tests following the standard BRRC protocol. The test substance was neither genotoxic nor cytotoxic to CHO cells under the conditions of these two in vitro test systems.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Apr 2012 - 20 Apr 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

only 2-AA was used as only positive control substance in presence of S9-mix.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Ä uvrB); ampicillin resistance (R factor plasmid).

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: E. coli WP2 uvrA was checked for UV sensitivity.

Metabolic activation:

with and without

Metabolic activation system:

cofactors supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats treated 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on 3 consecutive days

Test concentrations with justification for top dose:

1st Experiment (all strains, standard plate test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate
2nd Experiment (E.coli, standard test repeated with S-9 mix, due to inconclusive results in the first experiment; 3 plates/dose): 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate
3rd Experiment (all strains, preincubation test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2500; 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water.

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA), 4-nitro-o-phenylenediamine (NOPD), N-Methyl-N'-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

TEST DESIGN
Standard plate test (SPT) and preincubation test (PIT), both with and without metabolic activation, were performed.

Standard plate test (SPT, Experiment 1 and 2):
The experimental procedure was based on Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron & Ames (Mut. Res. 113: 173-215, 1983).
Test tubes containing 2 mL portions of soft agar [100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin for S. typhimurium or 0.5 mM tryptophan for E.coli)] were kept in a water bath at about 42 - 45°C. An amount of 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture and 0.5 mL S9 mix (in case of metabolic activation) or 0.5 mL phosphate buffer (in case of no metabolic activation) were added. After mixing, the samples were poured onto agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.

Preincubation Test (PIT, Experiment 3):
The experimental procedure was based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980). 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (in case of metabolic activation) or phosphate buffer (in case of no metabolic activation) were incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.

PARAMETERS EXAMINED
Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.

Titer:
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Cytotoxicity:
Toxicity was detected by (1) decrease in the number of revertants, (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth) and (3) reduction in the titer. Cytotoxicity was recorded for all test groups both with and without S9 mix in all experiments.

Solubility:
Precipitation of the test item was recorded and indicated. As long as precipitation does not interfere with colony scoring, 5000 µg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.

Evaluation criteria:

Acceptance criteria:
The experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain;
- The sterility controls reveales no indication of bacterial contamination;
- The positive control substances both with and without S9 mix induce a distinct increase in number of revertant colonies within the range of the historical positive control data or above;
- The titer of viable bacteria is egal to/greater than 10E+8/mL.

Assessment criteria:
The test item is positive if a dose-related and reproducible increase in the number of revertant colonies, i .e . about doubling of the spontaneous mutation rate in at least one tester strain either without or with S9-mix, is observed.
The test item is generally nonmutagenic if the number of revertants for all tester strains is within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease in the number of his+ revertants reported for TA 1537 without S9 mix at 2500 and 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY
When tested in the S. typhimurium strains TA1535, TA1537, TA100 and TA98 under standard plate test (SPT, Experiment 1) and preincubation test (PIT, Experiment 3) conditions, with and without S9 mix, the test item did not induce an increase in his+ revertant colonies at concentrations up to 5000 µg/plate.
For E. coli tested in the SPT in absence of S9 mix, no increase in trp+ revertant colonies at concentrations up to 5000 µg/plate was noticed. In contrast, when tested in presence of S9 mix, a slight increase in revertants was observed at the top dose of 5000 μg/plate, which however was not reproducible in a second standard plate test (Experiment 2) and in the preincubation test. Thus, the finding was regarded as not relevant.

CYTOTOXICITY
A cytototoxic effect was observed in the SPT in absence of S9 mix for TA 1537 only, from 2500 μg/plate onward.

PRECIPITATION
No test item precipitation was observed within the concentration range tested, with and without S9-mix.

CONTROLS
The number of revertant colonies in the negative controls was within the range of the historical negative control data reported in the study for each tester strain, both with and without S9-mix. The positive control substances induced the expected increased in revertant colonies, both with and without S9-mix; the results were within the historical positive control range. Thus, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

Remarks on result:

other: SPT Experiment 1

Triethylene glycol: Bacterial Reverse Mutation Assay
EXPERIMENT 1 (Standard Plate Test, SPT); mean revertants colonies/plate (mutation factor)
Strain	S. typhimurium strains
	TA1535			TA100			TA1537			TA98
Test item (µg/plate)	-S9		+S9	-S9	+S9		-S9		+S9	-S9	+S9
NC	10(1.0)*		12(1.0)	75(1.0)	92(1.0)		6(1.0)		7(1.0)	19(1.0)	22(1.0)
33	10(0.9)		13(1.1)	72(1.0)	93(1.0)		6(0.9)		6(1.0)	18(1.0)	23(1.0)
100	9(0.9)		11(0.9)	81(1.1)	98(1.1)C		6(0.9)		6(0.9)	18(1.0)	20(0.9)
333	9(0.9)		12(1.0)	77(1.0)	86(0.9)		6(0.9)		7(1.1)	18(0.9)	20(0.9)
1000	10(1.0)		13(1.1)	79(1.1)	86(0.9)		6(1.0)		7(1.1)C	17(0.9)	20(0.9)
2500	10(0.9)		12(1.0)C	83(1.1)	77(0.8)		5(0.7) (T)		5(0.8)	16(0.8)	19 (0.9)
5000	10(1.0)		12(1.0)	78(1.0)	86(0.9)		4(0.7) (T)		7(1.1)	14(0.8)	21(0.9)
PC	893(86.4)		211(17.6)	686(9.1)	783(8.5)		434(68.5)		172(25.8)	641(34.3)	754(33.8)
Strain	E.coli WP2uvrA			NC = negative (vehicle) control PC = respective positive control T = slight cytotoxicity C = contamination; only 2/3 plates evaluated
Test item (µg/plate)	-S9		+S9
NC	37(1.0)		41(1.0)
33	36(1.0)		42(1.0)
100	34(0.9)		50(1.2)
333	32(0.9)		52(1.3)
1000	37(1.0)		46(1.1)C
2500	34(0.9)		54(1.3)
5000	40(1.1)		65(1.6)
PC	873(23.6)		235(5.8)
EXPERIMENT 2 (Standard Plate Test, SPT); mean revertants colonies/plate (mutation factor)
E.coli WP2uvrA				NC = negative (vehicle) control PC = respective positive control
Test item (µg/plate)		+S9
NC		40(1.0)
33		37(0.9)
100		40 (1.0)
333		39(1.0)
1000		39(1.0)
2500		38(1.0)
5000		41(1.0)
PC		267 (6.7)
EXPERIMENT 3 (Preincubation Test, PIT);mean revertants colonies/plate (mutation factor)
Strain	S. typhimurium strains
	TA1535			TA100				TA1537		TA98
Test item (µg/plate)	-S9		+S9	-S9		+S9		-S9	+S9	-S9	+S9
NC	11(1.0)		13(1.0)	71(1.0)		84(1.0)		6(1.0)	7(1.0)	18(1.0)	25(1.0)
33	10(0.9)		13(1.0)	73(1.0)		79(0.9)		6(0.9)	7(1.1)	17(1.0)	23(0.9)
100	11(1.0)		11(0.9)	74(1.0)		94(1.1)		6(1.0)	7(1.1)	19(1.1)	24(1.0)
333	11(1.0)		13 (1.0)	75(1.1)		87(1.0)		7(1.1)	8(1.2)	18(1.0)	25(1.0)
1000	10(0.9)		12(0.9)	74(1.0)		93(1.1)		6(1.0)	8(1.2)	18(1.0)	22(0.9)
2500	11(1.0)		12(1.0)	80(1.1)		86(1.0)		5(0.8)	6(1.0)	18(1.0)	23(0.9)
5000	11(1.0)		11(0.9)	76(1.1)		82(1.0)		7(1.1)	7(1.1)	18(1.0)	24(1.0)
PC	670(60.9)		133(10.5)	875(12.3)		778(9.2)		356(56.3)	126(19.0)	587(32.6)	593(23.7)
Strain	E.coli WP2uvrA			NC = negative (vehicle) control PC = respective positive control
Test item (µg/plate)	-S9		+S9
NC	41(1.0)		47(1.0)
33	38(0.9)		49(1.0)
100	44(1.1)		41(0.9)
333	45(1.1)		46(1.0)
1000	47(1.1)		51(1.1)
2500	47(1.1)		56(1.2)
5000	48(1.2)		51(1.1)
PC	699(16.9)		242(5.1)

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Appearance: Slightly viscous; water-clear liquid
- Purity: 100%
- Specific gravity: 1.1254
- pH in a 50% solution: 7.51

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:

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:

triethylenemelamine

cyclophosphamide

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 invasion 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.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

- Appearance: Slightly viscous; water-clear liquid
- Purity: 100%
- Specific gravity: 1.1254
- pH in a 50% solution: 7.51

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:

24 - 50 mg/mL
Appropriate concentrations for mutagenicity testing were determined by preliminary measurements of cytotoxicity to CHO cells of rat-liver S9 metabolic activation system. Selection of a suitable range of concentrations for testing (0.003 - 50 mg/mL) was based upon an estimate of the doses which would not kill over 90% of the treated cells. The test substance showed not to be cytotoxic when tested up to a maximum of 50 mg/mL.

Vehicle / solvent:

Sterile water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

For determination of direct genotoxic action, CHO cells were exposed to the test substance 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" chromatid by SCE staining, was present at a concentration of 3 µg/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.

Statistics:

The data were analysed after transformation of the mutation frequencies MF) and SCE values according to the conversion method of Box and Cox (1964). For CHO mutation studied with a concurrent control frequency of zero mutants, the variance of recent historical controls was used for the statistical analyses.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance did not affect the incidence of SCEs over the range of concentrations tested with or without addition of an active S-9 metabolic activation system. No dose-related effects of exposure on the incidence of SCEs were evident and the test agent was inactive in the present in vitro assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro:

The test substance was tested for its mutagenic potential in Salmonella typhimurium and Escherichia coli, in the standard plate test (SPT) and the preincubation test (PIT), according to the OECD TG 471 (BASF 2012). The tester strains were: TA1535, TA100, TA1537, TA98 and E. coli WP2 uvrA. Testing was done in absence and presence of S9 mix. Negative and positive controls were included. The test concentrations were as follows:

Experiment 1 (all strains, standard plate test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Experiment 2 (E.coli, standard test repeated with S-9 mix, due to inconclusive results in the first experiment; 3 plates/dose): 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Experiment 3 (all strains, preincubation test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2500; 5000 µg/plate

When tested in all S. typhimurium strains mentioned above under SPT and PIT conditions, with and without S9 mix, the test item did not induce an increase in his+ revertant colonies at concentrations up to 5000 µg/plate. A cytotoxic effect was observed in the SPT in absence of S9 mix for TA 1537 only, at 2500 and 5000 μg/plate. For E. coli tested in the SPT in absence of S9 mix, no increase in trp+ revertant colonies at concentrations up to 5000 µg/plate was noticed. In contrast, when tested in presence of S9 mix, a slight increase in revertants (factor 1.6) was observed at the top dose of 5000 μg/plate, which however was not reproducible in a second standard plate test (Experiment 2) and in the preincubation test (Experiment 3). Thus, the finding was regarded as not relevant. No cytotoxic effect was noticed for E.coli. No test item precipitation was observed within the concentration range tested, with and without S9-mix. Referring to controls, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen, the test item triethylene glycol is not mutagenic in the bacterial reverse mutation assay in the absence and presence of metabolic activation.

 

In an in vitro mammalian chromosome aberration study in CHO cells (BRRC, 1986), the test substance did not produce increases in chromosome aberrations in comparison to control cultures with and without metabolic activation.

No evidence of clastogenic effects was recorded. Furthermore, negative results with and without metabolic activation were obtained with a sister chromatid exchange (BRRC, 1986).

A HGPRT assay with CHO cells tested in absence and presence of S9 mix was conducted with the read-across substance Diethylene glycol, equivalent or similar to OECD TG 476 (Union Carbide, 1984). 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. It was observed that diethylene glycol was not highly cytotoxic when tested either with or without S9 metabolic activation. A concentration of 50 mg/mL produced 16% inhibition of growth without S9 and 27% inhibition with S9. For the definitive tests, a concentration range between 30 to 50 mg/mL was tested in the mutagenicity tests with and without S9. In the main test, diethylene glycol was neither genotoxic nor cytotoxic to CHO cells under the conditions of this in vitro test system.

In vivo:

No data/studies are available. However, in the light of overall negative in vitro and in vivo results from genotoxicity experiments with other glycols and the negative in vitro results for triethylene glycol, classification and further studies are not needed.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available information classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.