2-(2-ethoxyethoxy)ethyl acetate

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:

From September 04, 2020 to December 02, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21, corrected 2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-ethoxyethoxy)ethyl acetate

EC Number:

203-940-1

EC Name:

2-(2-ethoxyethoxy)ethyl acetate

Cas Number:

112-15-2

Molecular formula:

C8H16O4

IUPAC Name:

2-(2-ethoxyethoxy)ethyl acetate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120
- Expiration date of the lot/batch: 2023-04-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Mitochondrial fraction from liver of male Sprague-Dawley rats treated with Aroclor 1254
- method of preparation of S9 mix: S9 fraction buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the S9 mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% v/v S9 mix for plate incorporation assay, 10% v/v S9 mix for pre-incubation assay
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes, by supplier.

Test concentrations with justification for top dose:

Plate incorporation assay: 0.0015 - 5 µLg/plate with and without metabolic activation
Pre-incubation assay: 0.1563 - 5 µLg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain TA1537 in the absence of S9

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains TA1535 and TA 100 in the absence of S9

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Strain TA98 in the absence of S9

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Strain TA102 in the absence of S9

Controls 5

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

All strains in the presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1: in medium; in agar (plate incorporation); Experiment 2: preincubation

DURATION
- Preincubation period: 30 min at 30 °C (only experiment 2)
- Exposure duration: 96 hours at 37 °C

NUMBER OF REPLICATIONS: triplicates per concentration in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants, reduction of background lawn


NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate/triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): ca 2 x 10E+9 CFU/mL
- Test substance added in medium; in agar (plate incorporation) (1st assay) and pre-incubation (2nd assay)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition (thinning of background lawn) and reduction in number of colonies

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Counting of numbers of revertant colonies

Rationale for test conditions:

As prescribed by test guidelines

Evaluation criteria:

The following criteria were used to determine a valid assay:
Tester Strain Integrity - All tester strain cultures exhibited sensitivity to crystal violet, demonstrating presence of the rfa wall mutation. All tester strains demonstrated a requirement for biotin except strain TA102 which is biotin independent. All tester strains showed sensitivity to UV exposure except TA102 which is wild type. All the tester strains demonstrated a requirement for histidine for their growth. Tester strains TA98, TA100 and TA102 exhibited resistance to ampicillin, demonstrating presence of the pKM101 plasmid. Tester strains TA102 exhibited resistance to tetracycline, demonstrating presence of the pAQ1 plasmid.

The following criteria were used to determine a positive result:
A result was considered positive if a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain, with or without metabolic activation, was seen.

Statistics:

Mean values and standard deviations were calculated. Simple linear regression analysis was performed fto assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: No
- Water solubility: Suitable for the assay
- Precipitation: No precipitation occurred up to the limit concentration of 5 µL/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Valid

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : None observed

Ames test:
- Signs of toxicity : Not observed
- Mean number of revertant colonies per plate and standard deviation : See tabulated data

- Positive historical control data: Test data fell within the historical control range
- Negative (solvent/vehicle) historical control data: Test data fell wihin the historical control range

Any other information on results incl. tables

Plate incorporation assay - Mean count of revertant colonies

 

Concentration (µL/plate)	His^+Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	7.00	2.83	14.00	2.83	19.00	0.00	122.00	5.66	206.00	2.83
0.0015	7.00	1.41	12.50	2.12	21.50	3.54	128.00	7.07	249.00	39.60
0.005	7.50	0.71	13.50	0.71	21.00	4.24	111.00	8.49	231.00	0.00
0.015	5.00	1.41	14.00	2.83	20.50	3.54	136.50	36.06	228.50	20.51
0.05	6.50	3.54	15.50	2.12	23.00	2.83	128.00	9.90	222.50	12.02
0.15	7.00	2.83	16.00	1.41	20.00	2.83	140.50	6.36	228.00	4.24
0.5	7.50	3.54	13.50	0.71	15.00	11.31	120.00	4.24	235.00	7.07
1.5	9.00	1.41	13.00	5.66	18.00	0.00	138.00	8.49	220.00	5.66
5	6.50	0.71	13.50	0.71	14.50	7.78	126.50	2.12	219.00	19.80
PC	173.00	63.64	307.00	55.15	460.00	29.70	682.50	55.86	927.50	101.12

Key: SD = Standard Deviation, NC = Negative Control, DW =Distilled Water, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate), 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100)},- = Not Applicable.

 

Plate incorporation assay - Mean count of revertant colonies

 

Concentration  (µL/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	8.50	0.71	16.50	4.95	25.00	4.24	137.50	3.54	225.50	21.92
0.0015	6.50	0.71	11.00	2.83	22.50	2.12	131.50	10.61	230.00	9.90
0.005	11.50	4.95	18.00	7.07	19.00	7.07	135.00	45.25	205.50	9.19
0.015	10.50	3.54	19.00	1.41	19.50	6.36	121.50	28.99	227.50	6.36
0.05	11.00	0.00	17.00	7.07	20.00	1.41	161.50	19.09	209.50	2.12
0.15	9.50	7.78	13.50	9.19	18.00	5.66	125.00	29.70	243.50	2.12
0.5	8.00	0.00	12.50	2.12	20.00	2.83	118.00	9.90	235.50	23.33
1.5	8.50	4.95	11.00	4.24	21.00	4.24	133.00	5.66	234.50	19.09
5	9.00	4.24	12.00	4.24	21.50	4.95	107.00	4.24	244.50	24.75
PC	242.50	3.54	270.00	12.73	676.50	94.05	688.50	78.49	947.50	99.70

  Key:     SD = Standard Deviation, NC = Negative Control, DW =Distilled Water, PC = Positive control {2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

Pre-incubation assay - Mean count of revertant colonies

 

Concentration (µL/plate)	His^+Revertant Colonies/Plate [Absence of Metabolic Activation]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	8.33	3.21	16.00	1.73	23.33	1.53	125.00	17.69	235.33	17.10
0.15625	8.67	1.15	15.33	3.06	26.33	3.06	134.33	13.05	234.33	7.37
0.3125	8.33	3.21	15.33	2.08	27.33	4.51	137.33	21.03	230.00	24.43
0.625	8.67	2.08	16.67	2.08	25.33	2.52	129.00	10.82	236.00	12.49
1.25	8.33	2.08	15.00	1.73	26.67	2.89	137.33	6.11	233.67	9.71
2.5	9.00	1.73	16.67	2.08	24.33	1.53	130.33	15.95	229.67	11.24
5	8.33	1.53	15.33	1.53	26.00	3.61	135.33	11.06	241.33	11.37
PC	188.33	16.01	282.00	11.36	570.67	72.60	695.33	17.79	1009.33	69.51
2Aa	-	-	-	-	-	-	122.33	11.06	-	-

Key: SD = Standard Deviation, NC = Negative Control, DW =Distilled Water,PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),- = Not Applicable.

Pre-incubation assay - Mean count of revertant colonies

 

Concentration (µL/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	9.33	2.52	16.00	2.00	27.33	2.52	127.00	8.00	228.00	20.95
0.15625	9.00	1.73	15.00	1.00	24.67	3.79	127.33	15.37	232.67	8.08
0.3125	7.00	2.65	15.33	3.06	27.33	1.53	133.67	9.61	236.67	12.42
0.625	7.67	0.58	16.00	2.65	26.67	2.52	130.67	10.02	238.00	16.09
1.25	9.33	3.06	14.67	2.08	25.33	4.04	127.67	10.50	230.33	9.71
2.5	7.67	2.08	17.00	1.00	27.33	3.79	137.67	12.22	228.67	19.43
5	8.00	1.00	15.67	2.52	25.33	2.08	134.00	13.23	237.00	14.00
PC	170.33	16.77	283.33	26.95	508.00	42.53	551.00	32.51	1126.67	163.52

Key:  SD = Standard Deviation, NC = Negative Control,DW =Distilled Water,PC = Positive Control{2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

Applicant's summary and conclusion

Conclusions:

The tested substance is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100 and TA102, when tested under the specified conditions.

Executive summary:

The mutagenic activity of 2 -(2 -ethoxyethoxy)ethyl acetate has been evaluated using the bacterial reverse mutation test, on five histidine deficient mutant tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102) using methods described by OECD test guidelines.

The substance did not induce any significant increase in the number of revertants, in trials with and without S9 mix, in any tester strain. Values for the negative control were within the historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.