2-[(2-ethylhexyl)oxy]ethanol

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-ethylhexyl)oxy]ethanol

EC Number:

216-323-7

EC Name:

2-[(2-ethylhexyl)oxy]ethanol

Cas Number:

1559-35-9

Molecular formula:

C10H22O2

IUPAC Name:

2-[(2-ethylhexyl)oxy]ethan-1-ol

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

Method

Species / strain

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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.

Applicant's summary and conclusion

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.