2-[(2-ethylhexyl)oxy]ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ethylene Glycol 2-Ethylhexyl Ether was not mutagenic/genotoxic under conditions used in these assays. Based on the key genotoxicity studies the chemical does not raise the cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physical state/Appearance: Clear colourless liquid
Batch: TXEG
Purity: 100%
Expiry Date: 08 February 2019
Storage Conditions: Approximately 4 °C in the dark

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus.The bacteria used in the test were obtained from: • University of California, Berkeley, on culture discs, on 04 August 1995
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Experiment 1 - Plate Incorporation Method

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method

Without and without Metabolic Activation- 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated.


Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count

Evaluation criteria:

Criteria for for determining a positive result

1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In the presence S9-mix, weakened bacterial background lawns were noted to Salmonella strains TA1535, TA98 and TA1537 at 5000 μg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the presence S9-mix, weakened bacterial background lawns were noted to Salmonella strains TA1535, TA98 and TA1537 at 5000 and 1500 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the presence S9-mix, weakened bacterial background lawns were noted to Salmonella strains TA1535, TA98 and TA1537 at 5000 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Small reductions in TA100 and TA1537 revertant colony frequency, without a weakening of the background lawn, were also noted at 1500 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix initially from 1500 μg/plate. In the presence S9-mix, weakened bacterial background lawns were noted to Salmonella strains TA1535, TA98 and TA1537 at 5000 μg/plate. Small reductions in TA100 and TA1537 revertant colony frequency, without a weakening of the background lawn, were also noted at 1500 μg/plate. Consequently, the maximum recommended dose (5000 μg/plate) was again selected as the maximum dose in the second mutation test. The test item induced a slightly stronger response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 μg/plate (TA1537) and 1500 μg/plate (TA100, TA1535, TA98 and WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 1500 μg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.
A light, greasy test item film was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

Conclusions:

Ethylene Glycol 2-Ethylhexyl Ether was considered to be non-mutagenic under the conditions of this test.

Executive summary:

A study was conducted following OECD TG No. 471 and EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test. The study was conducted in accordance with the GLP regulations.Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item (1.5 to 5000 μg/plate) using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without metabolizing system. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 2 (pre-incubation method). Ethylene Glycol 2-Ethylhexyl Ether was non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: Ethylene Glycol 2-Ethylhexyl Ether
CAS number: 1559-35-9
Physical state/Appearance: Clear colorless liquid
Purity: 100%
Expiry Date: 08 February 2019
Storage Conditions: Approximately 4 C in the dark
Intended use/Application: Solvent

Species / strain / cell type:

lymphocytes:

Remarks:

normal human lymphocytes

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours

Metabolic activation:

with and without

Test concentrations with justification for top dose:

4 (20)-hour without S9 and 4(20)-hour with S9 (2%)- 0, 60, 120, 240, 270, 300, 360, 420 μg/mL

24-hour without S9 0, 32, 64, 128, 192, 256, 384, 480 μg/mL

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 26 years
Main Experiment (24-hour only) male, aged 34 years
Main Experiment repeat (4(20)-hour exposures): female, aged 31 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No’s. PB/βNF S9 31/3/17 and PB/βNF S9 30/6/17 were used in this study. A copy of the S9 Certificate of Efficacy are presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: Human Lymphocytes

Remarks:

Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The dose range of test item used was 0, 60, 120, 240, 270, 300, 360 and 420 , 480 μg/mL.

Vehicle controls validity:

valid

Remarks:

All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes

Positive controls validity:

valid

Remarks:

All the positive control items induced statistically significant increases in the frequency of cells with aberrations

Conclusions:

The test item, Ethylene Glycol 2-Ethylhexyl Ether was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation at concentration range of 0- 480 μg/mL. The test item, Ethylene Glycol 2-Ethylhexyl Ether was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

The test substance is identified as Ethylene Glycol 2-Ethylhexyl Ether. The purity is 100%. The physical state/appearance of material is Clear colourless liquid. The expiry date of material is 08 February 2019. The substance can be stored at approximately 4C in the dark.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Group Concentration of Ethylene Glycol 2-Ethylhexyl Ether (μg/mL) plated for viability and mutant frequency
4-hour without S9 80, 100, 120, 140, 160, 180, 200
4-hour with S9 (2%) 80, 100, 120, 140, 160, 180, 200
24-hour without S9 15, 30, 60, 80, 100, 120

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:

1. The majority of the plates, for both viability (%V) and TFT resistance, are analysable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4 hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour exposure the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10-6 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10-6 mutant frequency per survivor are not acceptable and will be repeated.
5. Every test should also be evaluated as to whether the positive controls (EMS and CP) meets at least one of the following two acceptance criteria developed by the IWGT workgroup:
The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF.
The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10-6).
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.

Statistics:

Delta Building Monitoring System and Mutant 240C by York Electronic Research

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Test item precipitate was observed at and above 871.5 μg/mL in all three of the exposure groups at the end of the exposure periods

Vehicle controls validity:

valid

Remarks:

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

Positive controls validity:

valid

Remarks:

The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.One main Mutagenicity Test was performed. In this main test, L5178Y TK mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.The dose range of test item used in the main test was selected in range of 80 -200 μg/mL.The maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic/genotoxic potential of Ethylene Glycol 2-Ethylhexyl Ether has been characterized in bacterial and mammalianin vitromutagenicity tests. The potential mutagenicity of the test item was tested on thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell according to the OECD TG 490. Cells were treated with the test item at concentration up to 200 μg/mL both in the absence and presence of metabolic activation. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells and was non-mutagenic in this assay.In an in vitro chromosome aberration assay conducted according to OECD Guideline 473, there was no evidence of an increase in the number chromosomal aberrations or polyploidy in human lymphocytes cells in the presence or absence of metabolic activation system when treated with test item at concentration level of 0- 480 μg/mL. In a bacterial reverse mutation assay conducted according to EU Method B.13/14 and OECD Guideline 471, there was no increase in mutation frequency in any strain of Salmonella typhimurium or Escherichia coli at concentrations up to 5000 μg/plate in the presence or absence of metabolic activation. Ethylene Glycol 2-Ethylhexyl Ether was not mutagenic/genotoxic under conditions used in these key assays.

Justification for classification or non-classification

No in vitro or in vivo germ cell mutagenicity/genotoxicity studies were available. Based on the available key studies indicates that Ethylene Glycol 2-Ethylhexyl Ether is not expected to induce heritable mutations in the germ cells of humans and is not classified for “Mutagenicity/genotoxicity” according to GHS.