1-ethoxy-2-(2-methoxyethoxy)ethane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: September 28, 2021
End of experimental phase: October 11, 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The test item behaviour, solubility was not investigated after 24 hours incubation. This deviation is not considered to have affected the integrity and validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

1-ethoxy-2-(2-methoxyethoxy)ethane

EC Number:

213-690-5

EC Name:

1-ethoxy-2-(2-methoxyethoxy)ethane

Cas Number:

1002-67-1

Molecular formula:

C7H16O3

IUPAC Name:

1-ethoxy-2-(2-methoxyethoxy)ethane

Test material form:

liquid

Details on test material:

CAS No.: 1002-67-1
Molecular formula: C7H16O3
Molecular weight: 148.2 g/mol
Purity: >99.9 %
Appearance: water clear, liquid
Expiry date: 28 March 2023
Storage condition: at room temperature, protected from light

Specific details on test material used for the study:

Test item name: 1-ethoxy-2-(2-methoxyethoxy)ethane
Lot No.: 09511
CAS No.: 1002-67-1
Molecular formula: C7H16O3
Molecular weight: 148.2 g/mol
Purity: >99.9 %
Appearance: water clear, liquid
Expiry date: 28 March 2023
Storage condition: at room temperature, protected from light

Method

Target gene:

Thymidine Kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Supplier: American Type Culture Collection (ATCC) (Manassas,Virginia)
Product No.: CRL-9518
Lot No.: 63293486
Date of arrival: August 05, 2020
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection.
Cells are stored as frozen stocks (Master Copies: MC) in liquid nitrogen. Each batch of frozen cells is cleansed of pre-existing mutant cells. This is accomplished using methotrexate (aminopterin) to select against TK-deficient cells and adding thymidine, hypoxanthine and glycine (producing Master Products: MP). Each batch of cleansed cells is checked for the absence of mycoplasma for population doubling times and modal number of chromosomes. In the experiments the used MP batch (210506) is mycoplasma negative, has a doubling time of 9.41 hours (in the typical range of 8-11 hours) and a modal chromosome number of 40.
For the experiments 2 vials were thawed rapidly, the cells were diluted in RPMI 10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. When the cells are growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The test item was investigated in the presence of an appropriate metabolic activation system,
which is a cofactor-supplemented post-mitochondrial fraction (S9).
Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was
provided by Trinova Biochem GmbH, (Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA). Certificate of
Analysis was obtained from the supplier.
The Certificate of Analysis of rat liver S9 is stored in the Laboratory of TOXI-COOP ZRT.
The certified copies of the original certificates are collected in the raw data notebook of this
study.
The used S9 fractions:
Lot Number: 4303; Expiry date: August 27, 2022; Protein content: 37.6 mg/mL;
Lot Number: 4399; Expiry date: January 27, 2023; Protein content: 37.3 mg/mL.
5.6.2 The S9 Mix (with Rat Liver S9)
The S9 Mix was prepared as follows:
The concentration of the stock solutions for the S9 Mix:
NADP Na 1) 25 mg/mL
D-glucose-6 phosphate Na 180 mg/mL
KCl 150 mM
1) NADP= Nicotinamide-dinucleotide-phosphate
The complete S9 Mix (the ratio of the components is given referring to 10 mL of S9 Mix)
was freshly prepared immediately before each experiment containing components as
follows:
NADP Na stock solution 2 mL
D-glucose-6 phosphate Na stock solution 2 mL
KCl stock solution 2 mL
Rat liver homogenate (S9) 4 mL
The S9 Mix was kept in an ice bath until use.
For all cultures treated in the presence of S9 Mix, a 1 mL aliquot of the above S9 Mix was
added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the rat
liver homogenate in the test system was 2 %.
1 mL of 150 mM KCl was aliquoted to the cultures treated in the absence of S9 Mix.
In the main experiments the S9 Mix composition was exactly the same.
The details of the used chemicals (supplier/manufacturer, batch/lot number and expiry/retest
date) are summarized in "Any other information on materials"

Test concentrations with justification for top dose:

The test item concentrations were decided based on the results of the preliminary solubility and cytotoxicity tests and in accordance with the recommendation of cited OECD 490 Guideline.

For the main experiment the following 1-ethoxy-2-(2-methoxyethoxy)ethane concentration levels were chosen:
3-hour treatment (±S9): 1500, 500, 150, 50 and 15 µg/mL.
The top concentration of 1500 µg/mL corresponds to about 10 mM test item.

Vehicle / solvent:

The treatment medium (RPMI 5 Medium as an aqueous vehicle) was chosen as appropriate vehicle for preparing the test item solutions based on the information provided by the Sponsor (water solubility > 1 x 103 g/L); furthermore, on the basis of a non-GLP preliminary solubility trial experience.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Supplier: American Type Culture Collection (ATCC) (Manassas,Virginia)
Product No.: CRL-9518
Lot No.: 63293486
Date of arrival: August 05, 2020
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection.
Cells are stored as frozen stocks (Master Copies: MC) in liquid nitrogen. Each batch of frozen cells is cleansed of pre-existing mutant cells. This is accomplished using methotrexate (aminopterin) to select against TK-deficient cells and adding thymidine, hypoxanthine and glycine (producing Master Products: MP). Each batch of cleansed cells is checked for the absence of mycoplasma for population doubling times and modal number of chromosomes. In the experiments the used MP batch (210506) is mycoplasma negative, has a doubling time of 9.41 hours (in the typical range of 8-11 hours) and a modal chromosome number of 40.
For the experiments 2 vials were thawed rapidly, the cells were diluted in RPMI 10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. When the cells are growing well, subcultures were established in an appropriate number of flasks.

The study includes preliminary solubility and concentration range finding tests (cytotoxicity test) and a main mutation assay: a main experiment (main experiment I). In this study the short-term treatment yielded clear negative results and according to the OECD 490 Guideline there is no requirement for verification of a clearly positive or negative response.
Furthermore, the test item is not a nucleoside analogue and was adequately soluble at the examined concentrations; therefore, conducting a test with longer treatment (24-hours) was considered as not necessary for the final conclusion of the study.
Remark: The treatment and subculturing procedure were interrupted on the day 1 (September 29, 2021) in the presence of exogenous metabolic activation, because the cell concentrations and the appearance of cell suspensions did not allow further culturing. After revision of cell culturing conditions, chemicals, media, S9 components, contents and personal background, this part of the experiment was re-started (immediately on September 29, 2021). The obtained cell concentration data of interrupted part are not presented in this report.

Preliminary Experiments

The preliminary solubility test and preliminary cytotoxicity test were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of this test will be archived under the study code of present study.

Preliminary Solubility Investigations

The preliminary solubility investigations were performed for selection of the appropriate vehicle to be used in the subsequent experiments and investigation of test item behaviour in the applied test system.
The test item was dissolved in the treatment medium (RPMI 5 Medium) at the concentration of 50 mg/mL and further diluted accordingly. The behaviour of this solution was investigated in the applied test system. The observations are summarized in "Any other information on materials and methods".

Preliminary Cytotoxicity Test

A preliminary cytotoxicity test was performed for selection of the treatment concentrations to be used in the subsequent experiments: main experiment I. The test item was dissolved in RPMI 5 Medium and further diluted accordingly.
In the preliminary cytotoxicity test 3-hour treatments in the presence and absence of S9 (±S9) were performed to determine the test item toxicity. The treatment procedure of the cell cultures was the same as described below for the main experiment. In the preliminary cytotoxicity test, single cultures were used and positive controls were not included.
The treated cells were washed following treatment with RPMI 10 culture medium and resuspended in 10 mL RPMI 10 culture medium.
In the frame of the concentration range finding investigations the cell cultures were maintained in flasks for 2 days. During this culturing period, subculturing was performed daily. The cell density was adjusted to a concentration of 2×105/mL and transferred to flasks for further growth.
At the end of the incubation period (expression period, see below), the cell density, the suspension growth (SG) and relative suspension growth (RSG) values in the selected cultures were determined see "Any other information on materials and methods".

Main Experiment

Concentrations

Based on the test item solubility properties and the obtained cell growth data (in this case missing or equivocal cytotoxicity, slight changes within the biological variability range of the applied test system) and according to the referred guideline (OECD 490) 1500 µg/mL was chosen as highest test item concentration to be examined in the main experiment (main experiment I) in the absence and presence of exogenous metabolic activation (±S9).

According to the guideline: at test items where no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μL/mL, whichever is the lowest. The present test item has defined composition and an active component content of >99.9 % ; therefore, at the concentration choice the concentration of 10 mM was taken into consideration as top concentration, that corresponds to 1482 µg test item/mL (rounded up: 1500 µg/mL).
For the main experiment the following five 1-ethoxy-2-(2-methoxyethoxy)ethane concentration levels were chosen:
3-hour treatment (±S9): 1500, 500, 150, 50 and 15 µg/mL.

Treatment of the Cells

In the main experiment, 3-hour treatment was performed in the presence and absence of exogenous metabolic activation (±S9) see "Any other information on materials and mehods".
For the 3-hour treatment incubations, a portion of at least 1×107 cells were placed in each of a series of sterile flasks (with culturing surface of 75 cm2). The treatment medium contained a reduced serum level of 5 % (v/v) (RPMI 5).
A suitable volume of vehicle, test compound or positive control solution, and 1.0 mL of S9 Mix or of 150 mM KCl was added to a final volume of 20 mL per culture containing at least 1×107 cells. The treated concentrations and controls are summarized in "Any other information on materials and methods". Duplicate cultures were used for each treatment.
The solubility of the test item in the cultures was observed, noticed by the unaided eye at the beginning and end of treatment. Beside these observations the changes in pH and osmolality of the test media were followed.
After the 3-hour incubation at 37 °C ± 0.5 °C (approximately 5 % CO2 in air) with gentle shaking the cell cultures were centrifuged at 1200 rpm for 5 minutes, washed with 10 mL RPMI 10 Medium and suspended in 10 mL RPMI 10/tube. The cell densities, (due to sufficient number of survival cells), were adjusted to a concentration of 2×105/mL. Cells were transferred to flasks for growth through the expression period (in 20 mL of cell suspension).
In this study, based on the short-term treatments a test with longer treatment (24-hours) was considered not necessary for the final conclusion of the study.

Expression Period

The cultures were maintained in flasks for 2 days, and during this time the TK-/- mutation was expressed. During the expression period, subculturing was performed daily with the aim of not exceeding 1×106 cells per mL and, where possible maintaining sufficient number of cells consequently a sufficient number of spontaneous mutants during the expression phase. For this purpose, cell density was adjusted to a concentration of 2×105/mL and transferred to flasks for further growth.
From observations on recovery and growth of the cultures during the expression period, five test dose levels plus negative and positive controls (3-hour treatment, ±S9) were plated for cloning efficiency (viability) and for mutant selection (5-trifluorothymidine (TFT) resistance).

Plating for Cloning Efficiency (Viability)

At the end of the expression period, the cell density in the selected cultures was determined and adjusted to nominally 1×104/mL with RPMI 20 for plating for a cloning efficiency (viability) test. Samples from these cultures were diluted to 8 cells/mL as follows:
Initial Cell Concentration: 1 × 104/mL (A)
1. Dilution Step: 0.5 mL of Cell Suspension (A) + 9.5 mL of RPMI 10
Intermediate Cell Concentration: 5 × 102/mL (B)
2. Dilution Step: 0.8 mL of Cell Suspension (B) + 49.2 mL of RPMI 20
Final Cell Concentration: 8/mL
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microtiter plates (192 wells) resulting in an average of 1.6 cells per well. Microtiter plates were incubated at 37 ºC ± 0.5 °C containing approximately 5 % (v/v) CO2 in air for 10-11 days. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 5-trifluorothymidine (TFT) Resistance

At the end of the expression period, the cell concentrations were adjusted to 1×104/mL.
The TFT (300 µg/mL) was diluted 100-fold in the cell suspensions to give a final concentration of 3 µg/mL according to the followings: 89.1 mL of cell suspension + 0.9 mL of TFT.
Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microtiter plates (384 wells) resulting in average 2×103 cells per well. The microtiter plates were incubated at 37 ºC ± 0.5 °C containing approximately 5 % (v/v) CO2 in air for 10-11 days and wells containing clones were identified as above and counted.
In addition, scoring of large and small colonies was performed, as the additional information obtained may contribute to an understanding of the ability of the test chemical to cause point mutations and/or chromosomal events (mode of action).
For the applied microwell method small colony mutants are defined as those covering less than 25 % of the well’s diameter and large colony mutants as those that cover more than 25 % of the well’s diameter.
The number of wells containing large colonies and the number of wells containing small colonies were scored.
In this way small and large colony mutant frequencies (at the negative and positive controls) were also calculated.

Rationale for test conditions:

According to guideline

Evaluation criteria:

The cytotoxicity is defined as the Relative Total Growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2-day expression period and the Relative Cloning Efficiency (RCE) obtained at the time of mutant selection. The corresponding formulas are the follows:

Suspension Growth (SG1):

SG1 = Cell concentration on day1/Cell concentration on day 0

Suspension Growth (SG2):

SG2 = Cell concentration on day 2/concentration on day 1 *Cell

The total SG = SG1 × SG2

Relative Suspension Growth (RSG):

RSG = SG (test item)/SG (control)

In the applied microwell version for the calculation of the cloning efficiency, the viable counts (CV) is determined as the product of the total number of microwells (TW) and the probable number of colonies per well (P) on microwell plates:

CV=PV × TWv

From the zero term of the Poisson distribution the probable number of clones/well (P) on microtiter plates in which there are empty wells (EW, without clones) out of a total of wells (TW) is given by:

P = -ln (EW/TW)

Therefore, the cloning efficiency of viability plates (where TV is the total cell number per well):

CEV=CV / TV = (PV × TWV) / TV

The Relative Cloning Efficiency (RCE):

RCE = CEV (test) / CEV (control)]

The Relative Total Growth (RTG):

RTG = RCE × RSG

The mutant frequency (MF) is the cloning efficiency of mutant colonies in selective medium (CEM) adjusted by the cloning efficiency in non-selective medium at the time of mutant selection (CEV). It is calculated as:

MF = CEM /CEV.


CM = PM × TWM

CEM = CM / TM = (PM × TWM) / TM

Small and large colony mutant frequencies can be calculated in an identical manner, using the relevant number of empty wells for small and large colonies as appropriate.

Statistics:

An approach for defining positive and negative responses the biological relevance of the increased MF was considered first.
Instead of statistical analysis which is generally used, this test relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated the Global Evaluation Factor (GEF), which is based on the analysis of the distribution of the negative control. For this assay the GEF is 126 × 10-6 based on the respective OECD guidance (OECD 490) document.
However, as an additional analysis the distribution and the homogeneity of the obtained data within a given test was also determined and the statistical analysis of mutant frequencies (total wells with clones) was carried out by non-parametric Mann-Whitney U-Test by IBM® SPSS® Statistics, Version 25 (2017) statistical software program.
The data were checked for a linear trend in mutant frequency with treatment concentration using the adequate regression analysis.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Validity of the Mutation Assay

1.) Experimental conditions:
In this study two experimental conditions (short-term treatment in the absence and also in the presence of exogenous metabolic activation) were conducted in the main experiment (main experiment I). The results of the short-term treatments performed in the absence and presence of exogenous metabolic activation were clearly negative (±S9). The test item had adequate solubility in the applied test system (no precipitation was noticed in the cell cultures in the examined concentration range during the experiment); furthermore, the test item’s structural properties (the test item is not a nucleoside analogue) confirm the negative conclusion of this study without performance of a potential longer treatment, i.e., 24 hours without exogenous metabolic activation.
2.) Adequate number of concentrations are analyzed:
In the main experiment five test item concentrations were tested in the absence and also in the presence of exogenous metabolic activation (±S9). The chosen concentration ranges allowed the evaluation of each experimental part.
In the main experiment I there were five analyzable concentration levels at the 3-hour treatment in the absence (-S9) and in the presence of exogenous metabolic activation (+S9) where the highest concentration of 1500 µg/mL (±S9) was chosen based on the preliminary cytotoxicity test results, in accordance with the guideline criterion (OECD 490 Guideline [8]) for test items where no precipitate or limiting cytotoxicity is observed. At these test items the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μL/mL, whichever is the lowest. The present test item has defined composition and an active component content of >99.9 %; therefore, at the concentration choice the concentration of 10 mM was taken into consideration as top concentration, that corresponds to 1482 µg test item/mL (rounded up: 1500 µg/mL).
3.) Adequate number of cells are analyzed:
The treated and subcultured cell numbers were adequate to the aim of the study (see "Any other information on results", according to the referred OECD 490 Guideline [8]).
4.) For negative (vehicle) control cultures:
The mutant frequency falls within the 50-170 mutants per 106 viable cells range:
The actual values: Main Experiment at the 3-hour treatment (-S9): 76 ("Any other information on results");
Main Experiment at the 3-hour treatment (+S9): 106 ("Any other information on results");
The cloning efficiencies at the time of mutant selection falls within the 65-120 % range:
The actual values: Main Experiment at the 3-hour treatment (-S9): 107 % ("Any other information on results"); Main Experiment at the 3-hour treatment (+S9): 102 % ("Any other information on results");
The suspension growth is 8-32-fold at the 3-hour treatment:
The actual values: Main Experiment at the 3-hour treatment (-S9): 24 ("Any other information on results");
Main Experiment at the 3-hour treatment (+S9): 14 ("Any other information on results");
5.) For positive control chemicals:
The positive control RTG should not be less than 10 %:
In the Main Experiment, the RTG of the positive control chemical, 4-Nitroquinoline-Noxide (NQO): 49 %, the RTG of Cyclophosphamide (CP) was 14 % ("Any other information on results").
The positive control MF must be within the acceptable historical control data range, established in the laboratory:
The positive control MF values were in accordance with the acceptable historical control data ranges, established in the laboratory in all treatments ("Any other information on results").
The actual MF values: Main Experiment at the 3-hour treatment (NQO): 724 ("Any other information on results");
Main Experiment at the 3-hour treatment (CP): 967 ("Any other information on results");

The positive control chemicals had to meet at least one of the following two acceptance criteria: the positive controls should demonstrate an induced mutation frequency (IMF) of at least 300 x 10-6 above the spontaneous background MF, and at least 40 % of the IMF should be reflected in the small colony MF; or the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/vehicle control (a small colony IMF of 150 x 10-6).
In the Main Experiment the positive control induced mutant frequency (IMF) was higher than 300 × 10-6 in all cases; it was 648 at the 3-hour treatment (-S9), and 861 at the 3-hour treatment (+S9) (Table 10).
The 40 % of induced mutation frequencies did not reflect in the small colony MF in the absence of S9, but the positive control (CP) induced mutant frequency (IMF) was 861 ("Any other information on results"), and more than the 40 % of induced mutation frequencies (actually 344) reflected in the small colony MF (actually: 435), see "Any other information on results".
The small colony IMF was 174 in the absence of S9, and 403 in the presence of S9 (both higher than 150 x 10-6)), see "Any other information on results".

The results of the main experiment fulfilled all of the validity and acceptability criteria. The study was therefore considered to be fully valid.

10.2 Test Item Behaviour, pH, Osmolality

No precipitate or test item caused opalescence was observed in the treatment solutions immediately after the 3-hour treatment (±S9) even up to the highest concentrations.
The pH and osmolality values (±S9) were within acceptable ranges ("Any other information on results").
10.3 Results of the Main Experiment

In this experiment the cells were treated with a range of the test item concentrations for 3 hours in absence and also in presence of S9 Mix. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2×105/mL. Cells were transferred to flasks for growth through the expression period (for approximately 2 days). At the end of the expression period cells were allowed to grow and form colonies for about 1011 days in culturing plates with and without selective agent trifluorothymidine (TFT) for determination of mutations and cloning efficiency (viability).
In the main experiment the treatment duration was 3 hours and the treatments were performed in absence and also in presence of exogenous metabolic activation (±S9).
For the main experiment the following five 1-ethoxy-2-(2-methoxyethoxy)ethane concentration levels were chosen:
3-hour treatment (±S9): 1500, 500, 150, 50 and 15 µg/mL.
In the preliminary cytotoxicity test no precipitate or limiting cytotoxicity was observed; therefore in accordance with referred OECD 490 guideline the chosen highest test concentration was 10 mM (the present test item has defined composition and an active component content of >99.9 % (see details in Section 5.1.1)) that corresponds to 1482 µg test item/mL (rounded up: 1500 µg/mL).
3h treatment in the absence and presence of S9:
The obtained relative total growth (RTG) values remained almost in the same range as the concurrent vehicle control in the whole examined concentration range, in the absence and also in the presence of exogenous metabolic activation (±S9). The slightly lower (in the absence: 6-11 % lower than vehicle control RTG, in the presence: 9-30 % lower than the vehicle control RTG, not concentration-related changed in any case) RTG values were considered (even the higher: 30 % RTG values) rather as being within the biological variability range of the applied test system, than a test item effect.
The cloning efficiency data did not show any test item effect at the whole examined concentration range of 15-1500 µg/mL (±S9)("Any other information on results").
The obtained mutation frequencies remained nearly in the same range as the vehicle control at the whole examined concentration range of 15-1500 µg/mL (±S9). The obtained changes did not show concentration-related increasing tendency with increasing concentrations, and the calculated mutant frequencies remained far below the GEF criterion for a positive call in whole examined concentration range (±S9), "Any other information on results".
Furthermore, the mutant frequencies remained well within the laboratory’s historical control data range of the corresponding vehicle control (95 % confidence intervals of C-charts ("Any other information on results") and remained within the literature’s normal range (50-170 mutants per 106 viable cells, in accordance with OECD 490 Guideline [8]) at the whole examined concentration range in the absence and presence of exogenous metabolic activation (±S9).
The additional statistical analysis of mutation frequencies of test item treatments showed no statistically significant differences in comparison with the negative control in either case in the absence of exogenous metabolic activation.
The mutation rates of the test item treatments did not differ statistically significantly from that of the vehicle control in most cases, in the presence of exogenous metabolic activation (Mann Whitney U-Test), "Any other information on results", but the mutation frequency differed statistically significantly from that of the vehicle control at 150 µg/mL concentration, in the presence of exogenous metabolic activation (Mann-Whitney U-test) (see "Any other information on results").
The statistically significant difference due to its sporadic appearance and intensity (the slightly higher mutation rate at this concentration level remained well within the laboratory’s historical control data range, see above) was considered rather as being within the biological variability range of the applied test system than test item effect.
The results of main experiment are presented in the "Any other information on results"

Any other information on results incl. tables

Summary Table of the Main Experiment

Suspension Concentration Growth (µg/mL) (SG)		Relative Total Growth (% RTG)	Cloning Efficiency (CEV)	Mutant  frequency (MF) (× 10^-6)	Threshold for positivity based on Global Evaluation Factor (GEF) ( × 10^-6)
3-hour treatment (-S9 Mix)					76 + 126 = 202
RPMI 5 Medium	24	100	107	76
15	23	89	96	71
50	24	94	101	81
150	22	90	104	82
500	19	90	117	98
1500	20	91	114	80
NQO (0.2 µg/mL)	14	49	91	724
3-hour treatment (+S9 Mix)					106 + 126 = 232
RPMI 5 Medium	14	100	102	106
15	11	91	119	90
50	10	76	108	118
150	10	70	100	132
500	11	74	97	117
1500	12	87	107	107
CP (5 µg/mL)	4	14	50	967

 

SG, RSG and RTG Data of the Main Experiment

Concentration (µg/mL)	Suspension growth (SG)	Relative Suspension Growth (% RSG)	Relative Total Growth (% RTG)
Treatment period (hours): 3
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium  (Vehicle Control)	23.67	100.00	100.00
15	23.42	98.96	88.80
50	23.72	100.22	94.34
150	22.09	93.34	90.31
500	19.44	82.13	89.65
1500	20.17	85.22	90.76
Positive reference control (NQO) (0.2 µg/mL) #	13.63	57.60	48.74
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium  (Vehicle Control)	14.28	100.00	100.00
15	11.07	77.48	90.57
50	10.34	72.41	76.30
150	10.19	71.34	70.26
500	11.13	77.94	74.41
1500	11.91	83.40	87.35
Positive reference control (CP) (5 µg/mL)	3.99	27.91	13.61

NQO         :  4-Nitroqinoline-N-oxide

CP            :    Cyclophosphamide

Remarks: The formulas for calculation of suspension growth, relative suspension growth and relative total growth values are detailed in the Section 7.1.

 #:  In this experimental part of the study, the positive NQO control values were compared with the RPMI 5 Medium vehicle control values. Additional dimethyl sulfoxide (DMSO) control as vehicle control for the positive control item NQO was not examined.

Summarized Results of the Cloning Efficiency (Viability) Data of the Main Experiment

Concentration (µg/mL)	Number of empty wells/  total number of wells	Cloning Efficiency (CEv)	Relative Cloning Efficiency (% RCE)
Treatment period (hours): 3
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium  (Vehicle Control)	65/384	107.13	100.00
15	78/384	96.14	89.74
50	72/384	100.85	94.13
150	71/384	103.66	96.76
500	55/384	116.94	109.15
1500	58/384	114.09	106.50
Positive reference control (NQO) (0.2 µg/mL) #	87/384	90.66	84.63
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium  (Vehicle Control)	72/384	102.03	100.00
15	54/384	119.27	116.89
50	65/384	107.51	105.37
150	72/384	100.48	98.48
500	76/384	97.40	95.46
1500	65/384	106.86	104.74
Positive reference control (CP) (5 µg/mL)	168/384	49.77	48.78

NQO :  4-Nitroqinoline-N-oxide

CP        :    Cyclophosphamide

Remarks: The formulas for calculation of cloning efficiency and relative cloning efficiency are detailed in the Section 7.1.

 #:  In this experimental part of the study, the positive NQO control values were compared with the RPMI 5 Medium vehicle control values. Additional dimethyl sulfoxide (DMSO) control as vehicle control for the positive control item NQO was not examined.

 

Summarized Mutagenicity Results of the Main Experiment

Concentration (µg/mL)	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Mutant  frequency (MF) (× 10^-6)	Small colony MF (× 10^-6)	Large colony MF (× 10^-6)
Treatment period (hours): 3
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium  (Vehicle Control)	649/768	94/768	25/768	75.70	14.90	58.64
15	666/768	79/768	23/768	71.42	15.29	54.24
50	648/768	86/768	34/768	81.27	21.66	56.70
150	644/768	84/768	37/768	81.71	23.02	55.96
500	606/768	124/768	38/768	98.29	20.96	72.65
1500	635/768	103/768	30/768	80.45	16.89	60.80
Positive reference control (NQO) (0.2 µg/mL)	198/768	340/768	230/768	723.65 ** IMF: 647.95 40 % of IMF = 259.18	189.20 Small colony IMF: 174.31	310.13 Large colony IMF: 251.49
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium  (Vehicle Control)	613/768	104/768	51/768	106.47	32.46	68.61
15	615/768	121/768	32/768	89.93	17.16	69.35
50	590/768	132/768	46/768	118.28	27.66	84.49
150	583/768	119/768	66/768	132.18 *	43.02	80.61
500	608/768	116/768	44/768	116.54	29.22	81.39
1500	607/768	111/768	50/768	106.84	30.41	70.51
Positive reference control (CP)  (5 µg/mL)	285/768	205/768	278/768	967.20 ** IMF: 860.73 40 % of IMF = 344.29	435.42 Small colony IMF: 402.96	300.65 Large colony IMF: 232.03

NQO :  4-Nitroqinoline-N-oxide

CP        :    Cyclophosphamide

IMF        :              Induced Mutant Frequency (increase in MF above concurrent control) **        :              Statistically significant, Mann-Whitney U Test; α=0.01. 

*           :    Statistically significant, Mann-Whitney U Test; α=0.05. 

 

Remark: No statistical analysis of small and large colony MF values was performed.

 

 

Cell Concentrations in the Main Experiment

Test solution (concentration  in µg/mL)	Post treatment cell concentrations (cell/mL)	Cell concentration during the expression period  (day 1) (cell/mL)	Cell concentration at the end of the expression period (day 2) (cell/mL)
Treatment period (hours): 3
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium (Vehicle Control)	5 8.47 × 10	1.64 × 10 ^6	2.32 × 10 ^6
15	8.97 × 10 ^5	1.62 × 10 ^6	2.31 × 10 ^6
50	8.63 × 10 ^5	1.59 × 10 ^6	2.38 × 10 ^6
150	8.83 × 10 ^5	1.56 × 10 ^6	2.26 × 10 ^6
500	8.83 × 10 ^5	1.59 × 10 ^6	1.96 × 10 ^6
1500	9.02 × 10 ^5	1.60 × 10 ^6	2.02 × 10 ^6
Positive reference control (NQO) (0.2 µg/mL)	8.92 × 10 ^5	1.43 × 10 ^6	1.53 × 10 ^6
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium (Vehicle Control)	5 6.27 × 10	1.06 × 10 ^6	2.16 × 10 ^6
15	6.50 × 10 ^5	9.78 × 10 ^5	1.81 × 10 ^6
50	6.77 × 10 ^5	9.03 × 10 ^5	1.83 × 10 ^6
150	6.95 × 10 ^5	8.97 × 10 ^5	1.82 × 10 ^6
500	6.75 × 10 ^5	9.62 × 10 ^5	1.85 × 10 ^6
1500	6.88 × 10 ^5	9.85 × 10 ^5	1.94 × 10 ^6
Positive reference control (CP) (5 µg/mL)	7.05 × 10 ^5	7.17 × 10 ^5	8.90 × 10 ^5

NQO      :  4-Nitroqinoline-N-oxide

CP           : Cyclophosphamide

Remark: The cultures after washing, after centrifuging (post treatment, day 1, day 2) were re-suspended in 10 mL RPMI 10/tube.

 

Individual Cloning Efficiency (Viability) Data of the Main Experiment

Test Item:		1-ethoxy-2-(2-methoxyethoxy)ethane
Date of Treatment:		−S9: September 28, 2021; +S9: September 29, 2021
Treatment Duration:		3 h (±S9 Mix)
Date of Plating for Viability:		−S9: September 30, 2021; +S9: October 01, 2021
Evaluation of Plates:		October 11, 2021
Without Exogenous Metabolic Activation (-S9 Mix); Treatment Duration: 3h
Concentration (µg/mL)			Number of Empty Wells
	Parallels:		A			B
			1	2	1		2
RPMI 5 Medium  (Vehicle Control)			13	16	17		19
15			16	23	20		19
50			16	16	21		19
150			24	21	14		12
500			12	14	13		16
1500			15	11	16		16
Positive reference control (NQO) (0.2 µg/mL)			19	31	18		19
With Exogenous Metabolic Activation (+S9 Mix); Treatment Duration: 3h
Concentration (µg/mL)			Number of Empty Wells
	Parallels:		A			B
			1	2	1		2
RPMI 5 Medium  (Vehicle Control)			18	23	19		12
15			11	17	10		16
50			18	13	14		20
150			19	17	18		18
500			17	21	18		20
1500			15	15	19		16
Positive reference control (CP) (5 µg/mL)			44	38	46		40

NQO :  4-Nitroqinoline-N-oxide

CP          : Cyclophosphamide

 

Individual Mutagenicity Data of the Main Experiment

Test Item:		1-ethoxy-2-(2-methoxyethoxy)ethane
Date of Treatment:		−S9: September 28, 2021; +S9: September 29, 2021
Treatment Duration:		3 h (±S9 Mix)
Date of Plating for Mutagenicity:		−S9: September 30, 2021; +S9: October 01, 2021
Evaluation of Plates:		October 11, 2021
Without Exogenous Metabolic Activation (-S9 Mix), Treatment Duration: 3h
Concentration (µg/mL)			Number of Empty Wells
	Parallels:		A						B
			1	2	3	4	1	2		3	4
RPMI 5 Medium  (Vehicle Control)			76	79	82	83	85	81		84	79
15			87	87	83	84	83	32		78	82
50			84	86	81	79	84	76		81	77
150			86	81	79	79	81	79		80	79
500			74	81	77	70	66	81		75	82
1500			83	78	86	79	76	75		82	76
Positive reference control (NQO) (0.2 µg/mL)			30	25	20	24	29	20		22	28
With Exogenous Metabolic Activation (+S9 Mix); Treatment Duration: 3h
Concentration (µg/mL)			Number of Empty Wells
	Parallels:		A						B
			1	2	3	4	1	2		3	4
RPMI 5 Medium  (Vehicle Control)			76	81	80	76	75	71		80	74
15			78	79	75	84	75	71		73	80
50			73	76	69	73	68	74		78	79
150			71	68	71	74	70	77		79	73
500			79	79	88	73	71	69		71	78
1500			82	64	80	74	78	75		74	80
Positive reference control (CP) (5 µg/mL)			40	31	40	25	36	41		38	34

NQO :  4-Nitroqinoline-N-oxide

CP          : Cyclophosphamide

 

Individual Mutagenicity Data of the Main Experiment (The Number of Large and Small Colonies)

Test Item:								1-ethoxy-2-(2-methoxyethoxy)ethane
Date of Treatment:								September 28, 2021
Treatment Duration:								3 h (−S9 Mix)
Date of Plating for Mutagenicity:								September 30, 2021
Evaluation of Plates:								October 11, 2021
Without Exogenous Metabolic Activation (-S9 Mix); Treatment Duration: 3h
Concentration (µg/mL)							Number of large and small colonies
	Parallels:	A													B
		1		2			3		4		1			2		3			4
		LC	SC	LC	SC	LC		SC	LC	SC	LC	SC	LC	SC	LC		SC	LC	SC
RPMI 5 Medium  (Vehicle Control)		14	6	15	2	9		5	11	2	10	1	13	2	9		3	13	4
15		8	1	8	1	11		2	10	2	11	2	10	4	11		7	10	4
50		10	2	7	3	11		4	13	4	10	2	15	5	9		6	11	8
150		6	4	7	8	10		7	15	2	13	2	10	7	14		2	12	5
500		18	4	13	2	15		4	19	7	21	9	13	2	15		6	10	4
1500		10	3	16	2	8		2	14	3	15	5	12	9	12		2	16	4
Positive reference control (NQO) (0.2 µg/mL)		40	26	47	24	44		32	39	33	42	25	45	31	41		33	42	26

NQO :  4-Nitroqinoline-N-oxide

    SC       :  Small colony

    LC       :  Large colony

 

Individual Mutagenicity Data of the Main Experiment (The Number of Large and Small Colonies) (continued)

Test Item:							1-ethoxy-2-(2-methoxyethoxy)ethane
Date of Treatment:							September 29, 2021
Treatment Duration:							3 h (+S9 Mix)
Date of Plating for Mutagenicity:							October 01, 2021
Evaluation of Plates:							October 11, 2021
With Exogenous Metabolic Activation (+S9 Mix); Treatment Duration: 3h
Concentration (µg/mL)		Number of large and small colonies
	Parallels:	A												B
		1		2		3		4		1			2		3			4
		LC	SC	LC	SC	LC	SC	LC	SC	LC	SC	LC	SC	LC		SC	LC	SC
RPMI 5 Medium  (Vehicle Control)		15	5	9	6	13	3	11	9	13	8	16	9	12		4	15	7
15		13	5	13	4	15	6	10	2	17	4	20	5	20		3	13	3
50		16	7	17	3	21	6	15	8	21	7	15	7	13		5	14	3
150		16	9	16	12	17	8	15	7	18	8	12	7	11		6	14	9
500		11	6	12	5	7	1	15	8	18	7	22	5	17		8	14	4
1500		9	5	20	12	11	5	17	5	11	7	14	7	16		6	13	3
Positive reference control (CP) (5 µg/mL)		21	35	28	37	27	29	30	41	28	32	25	30	19		39	27	35

    CP       :  Cyclophosphamide

    SC       :  Small colony

    LC       :  Large colony

 

Mutant Frequency Historical Control Data

	Mutant Frequency / 10^6 Cells  (3-hour treatment without S9)
	RPMI 5 Medium Control	4-Nitroqinoline-N-oxide (NQO) Positive Control
Mean	89.51	796.35
Lower confidence interval	50.29	594.25
Upper confidence interval	128.73	998.46
SD	18.86	98.11
n	22	25
	Mutant Frequency / 10^6 Cells  (3-hour treatment with S9)
Treatments	RPMI 5 Medium Control	Cyclophosphamide (CP) Positive Control
Mean	96.72	1228.17
Lower confidence interval	56.91	812.44
Upper confidence interval	136.53	1643.91
SD	19.32	201.81
n	25	25

n: number of experiments

SD: Standard deviation

 

pH and Osmolality Data of the Main Experiment

Concentration (µg/mL)	pH	Osmolality (mmol/kg)
Treatment period (hours): 3
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium  (Vehicle Control)	7.68	321
15	7.71	302
50	7.71	303
150	7.72	304
500	7.70	305
1500	7.70	313
Positive reference control (NQO) (0.2 µg/mL)	7.73	459
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium  (Vehicle Control)	7.55	310
15	7.57	309
50	7.57	307
150	7.56	310
500	7.58	310
1500	7.56	311
Positive reference control (CP) (5 µg/mL)	7.58	308

NQO: 4-Nitroqinoline-N-oxide; CP: Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test item 1-ethoxy-2-(2-methoxyethoxy)ethane was clearly negative, did not induce gene mutations in presence and absence of metabolic activation in cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line).

Executive summary:

Purpose of the study:

An in vitro mammalian cell gene mutation assay was performed in mouse lymphoma L5178Y TK^+/- 3.7.2 C cells at the tk locus to test the potential of 1-ethoxy-2-(2methoxyethoxy)ethane to cause gene mutation and/or chromosome damage.

General procedures:

In the performed mutation assay the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2×10⁵/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days). At the end of the expression period the cells were allowed to grow and form colonies for approximately 10-11 days in culturing plates with and without selective agent trifluorothymidine (TFT) for determination of mutations and cloning efficiency (viability). 

In the main assays the suspension growth, the relative suspension growth, the relative total growth of the cells, the cloning efficiency (viability: colony-forming ability at the end of the 2-day expression period following the treatment) and the potential mutagenicity (5trifluorothymidine resistance) were determined.

Experimental phases: 

The study included a preliminary solubility trial, a concentration range finding test (cytotoxicity test) and a main mutation assay: a main experiment (main experiment I) (3hours treatment in the absence and presence of exogenous metabolic activation, ±S9). In this study the short-term treatment yielded clear negative results and according to the OECD 490 Guideline there is no requirement for verification of a clearly positive or negative response. 

Furthermore, the test item is not a nucleoside analogue and was adequately soluble at the examined concentrations; therefore, conducting a test with longer treatment (24-hours) was considered as not necessary for the final conclusion of the study.

Vehicle, test item concentrations, rationale for concentration selection: 

Based on the results of the preliminary solubility and toxicity tests, the test item was dissolved and diluted in RPMI 5 Medium (in the treatment medium). The RPMI 5 Medium was parallel investigated as vehicle control. Evident that the RPMI 5 Medium (as treatment medium) has no deleterious effect on the test system.

For the main experiment the following five 1-ethoxy-2-(2-methoxyethoxy)ethane concentration levels were chosen: 

3-hour treatment (±S9): 1500, 500, 150, 50 and 15 µg/mL.

In the preliminary cytotoxicity test no precipitate or limiting cytotoxicity was observed; therefore, in accordance with referred OECD 490 guideline the chosen highest test concentration was 10 mM (the present test item has defined composition and an active component content of >99.9 %) that corresponds to 1482 µg test item/mL (rounded up: 1500 µg/mL). 

Solubility, precipitation: 

No precipitate or test item caused opalescence was observed in the treatment solutions immediately after treatment, and after the 3-hour treatment even up to the concentration of 1500 µg/mL.

The pH and osmolality values remained within acceptable ranges throughout the study. 

Cytotoxicity results: 

In this study no cytotoxicity was noticed. The slight changes in the relative total growth (RTG) values were considered as being within the biological variability range of the applied test system.

Mutagenicity results: 

The experiments fulfilled the validity criteria regarding the parallel investigated negative control and positive control treatments, the number of analyzable concentration levels (at least four), analyzed cell numbers and chosen top concentration (10 mM, no precipitate or limiting cytotoxicity occurred in the preliminary experiments). 

In the present Mouse Lymphoma Assay (MLA), the short-term treatment (main experiment I) yielded clearly negative results in the absence and also in the presence of exogenous metabolic activation (±S9).

The obtained mutation frequencies remained nearly in the same range as the vehicle control at the whole examined concentration range of 15-1500 µg/mL (±S9). The obtained changes did not show concentration-related increasing tendency with increasing concentrations, and the calculated mutant frequencies remained far below the GEF (Global Evaluation Factoraccording to OECD 490 Guideline) criterion for a positive call in whole examined concentration range (±S9).

Furthermore, all of the obtained the mutant frequencies (±S9) of test item treatments remained well within the laboratory’s historical control data range of the corresponding vehicle control (95 % confidence intervals of C-charts) and within the literature’s normal range (50-170 mutants per 10⁶ viable cells, in accordance with OECD 490 Guideline [8]). 

The additional statistical analysis of mutation frequencies confirmed the clearly negative mutagenicity results, no statistically significant differences were obtained in most of the test item treatment-vehicle control comparisons (±S9), but the mutation frequency differed statistically significantly from that of the vehicle control at 150 µg/mL concentration, in the presence of exogenous metabolic activation (Mann-Whitney U-test). This latter change was considered as being unequivocally the biological variability of the applied test system. 

 

Under the conditions of this study, the test item 1-ethoxy-2-(2-methoxyethoxy)ethane was clearly negative, did not induce gene mutations in presence and absence of metabolic activation in cultured mammalian cells (L5178Y TK^+/- 3.7.2 C mouse lymphoma cell line).