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Administrative data

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 chromosome aberration assay, a sister chromatid exchange assay and an HGPRT gene mutation assay.
All available in vitro data revealed that the test item is not a mutagenic/genotoxic substance.

Link to relevant study records

Referenceopen allclose all

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:
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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to
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)

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
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.
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:
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 and an HGPRT gene mutation assay (BRRC, 1986).

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.