2-(2-ethoxyethoxy)ethyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria - Negative

Gene mutation in mammalian cells - Negative

Cytogenicity - Negative

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:

From September 04, 2020 to December 02, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

Target gene:

his operon

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain TA1537 in the absence of S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains TA1535 and TA 100 in the absence of S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Strain TA98 in the absence of S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Strain TA102 in the absence of S9

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.

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

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

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

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

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

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)}.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 17, 2020 to February 11, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum Essential Medium, Eagle α-Modification with/without nucleosides
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (Aroclor 1254 induced male SD rat liver ).

Test concentrations with justification for top dose:

Concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL investigated, the highest concentration examined being that recommended by the OECD test guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvent (water)
- Justification for choice of solvent/vehicle: Selected solvent is compatible with the test system

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

-S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: single

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2E+5/mL
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 8 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time (if incubation with a selective agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days
- Selective agent: 6-thioguanine, 5 µg/mL, 8 days cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2E+5 cells/mL seeded. Enumeration of cells by counting following staining

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

In accordance with OECD test guideline

Evaluation criteria:

The test item was considered to be positive if:
- At least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- The increase was concentration-related when evaluated with an appropriate trend test.
- Any of the results were outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit)

The test item was considered negative if:
- None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- There was no concentration-related increase when evaluated with an appropriate trend test.
- All results were inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit).

Statistics:

Kastenbaum MA and Bowman KO: Tables for determining the statistical significance of mutation frequencies. Mutation Research 9 527-549 1970.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: good
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: yes - concentrations of 0.063, 0.125, 0.25, 0.5, 1 and 2 µL/mL

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY : N/A

Summary of Absolute and Relative Cloning Efficiency at selection

 

Group (µL/mL)	Absence of Metabolic Activation			Presence of Metabolic Activation (2% v/v S9 mix)
	Mean ACE	RCE	RCE (%)	Mean ACE	RCE	RCE (%)
NC (DW)	0.6419	1	100	0.6536	1.0000	100.00
T1 (0.125)	0.6339	0.9876	98.76	0.5730	0.8767	87.67
T2 (0.25)	0.6441	1.0034	100.34	0.5858	0.8963	89.63
T3 (0.5)	0.6607	1.0294	102.94	0.6383	0.9767	97.67
T4 (1)	0.6140	0.9566	95.66	0.5822	0.8908	89.08
T5 (2)	0.6123	0.9539	95.39	0.6052	0.9259	92.59
PC	0.6166	0.9607	96.07	0.5765	0.8821	88.21

Key: NC = Negative control, DW = Distilled water, ACE = Absolute cloning efficiency, RCE = Relative cloning efficiency, T = Treatment concentration, PC = Positive control [0.4 µL Ethyl Methanesulfonate/mL in absence of metabolic activation and 6 µg Benzo (a) pyrene/mL in presence of metabolic activation]

Mutant Frequency per 1 x 10⁶Clonable Cells

 

Group (µL/mL)	Absence of Metabolic Activation						Presence of Metabolic Activation (2% v/v S9 mix)
	R	ACE	N° CP	N° CC	N° MC	MF	ACE	N° CP	N° CC	N° MC	MF
NC (DW)	1	0.6064	2439660	1479410	31	20.95	0.6750	2439360	1646568	34	20.65
	2	0.6773	2440140	1652707	30	18.15	0.6321	2439360	1541919	29	18.81
T1 (0.125)	1	0.6071	2439360	1480935	26	17.56	0.5821	2440320	1420510	30	21.12
	2	0.6607	2439360	1611685	31	19.23	0.5638	2439360	1375311	28	20.36
T2 (0.25)	1	0.6321	2439564	1542048	32	20.75	0.6179	2439360	1507281	29	19.24
	2	0.6560	2439684	1600433	28	17.50	0.5536	2439564	1350543	27	19.99
T3 (0. 5)	1	0.6750	2439600	1646730	29	17.61	0.6064	2439480	1479301	28	18.93
	2	0.6464	2439564	1576934	30	19.02	0.6702	2439660	1635060	31	18.96
T4 (1)	1	0.5851	2439480	1427340	32	22.42	0.5643	2439840	1376802	29	21.06
	2	0.6429	2440200	1568805	22	14.02	0.6000	2439996	1463998	20	13.66
T5 (2)	1	0.5709	2440140	1393076	32	22.97	0.6429	2439720	1568496	33	21.04
	2	0.6536	2440236	1594938	32	20.06	0.5674	2439360	1384093	27	19.51
PC	1	0.5857	2440104	1429169	557	389.74	0.5816	2440524	1419409	511	360.01
	2	0.6475	2439840	1579796	468	296.24	0.5714	2440476	1394488	473	339.19

Key: NC = Negative control, DW = Distilled water, ACE = Absolute cloning efficiency, N° CP = N° of cells plated, N° CC = N° of clonable cells, N° MC = N° of mutant colonies, MF = Mutant frequency per 10⁶clonable cells, T = Treatment concentration, PC = Positive control [0.4 µL Ethyl methanesulfonate/mL in absence of metabolic activation and 6 µg Benzo(a)pyrene/mL in presence of metabolic activation]

Conclusions:

2 -(2 -ethoxyethoxy) ethyl acetate is considered to be non mutagenic in the CHO/HGPRT forward mutation assay

Executive summary:

The potential for 2 -(2 -ethoxyethoxy) ethyl acetate to cause gene mutations in a mammalian cell line has been investigated using methods in accordance with OECD test guideline 476.

The results from the study demonstrate that 2 -(2 -ethoxyethoxy) ethyl acetate does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells either in the absence or presence of a metabolic activation system

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 17, 2020 to March 01, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human blood lymphocytes
- Sex, age and number of blood donors: Single male 28 years of age
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: n/a

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI-1640 medium, 37 deg C, 5% CO2

Cytokinesis block (if used):

Cytochalasin B

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: 2% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes, by supplier.

Test concentrations with justification for top dose:

0.0 (control), 0.125, 0.25, 0.5, 1.0 and 2 µL/mL
Highest tested concentration as recommended by test guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvent (water)
- Justification for choice of solvent/vehicle: Selected solvent is compatible with the test system

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

vinblastine

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: yes

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Short-term treatment - 4 hours; Long-term treatment - 24 hours
- Harvest time after the end of treatment (sampling/recovery times): 24 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Identity of cytokinesis blocking substance - cytochalasin B; Concentration - 6 µg/mL; Duration and period of cell exposure - 24 hours
- Methods of slide preparation and staining technique used including the stain used: Slides were prepared from fixed cell suspension placed drop by drop onto pre-cleaned, ice-chilled slides and then dried over a slide warmer. Dried slides were stained with 5% Giemsa solution for 30 minutes
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 x replicates, 1000 cells/replicate
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Criteria for selecting cells suitable for scoring Micronuclei - The cytokinesis-blocked binucleated cells that were selected for scoring (for MN frequency) had the following characteristics:
• The cells were binucleated.
• The two nuclei in a binucleated cell had intact nuclear membranes and situated within the same cytoplasmic boundary.
• The two nuclei in a binucleated cell were approximately equal in size, staining pattern and staining intensity.
• The two nuclei within a BN cell whenever attached by a nucleoplasmic bridge, it was no wider than 1/4th of the nuclear diameter.
• The two main nuclei in a BN cell might touch but ideally they were not overlapping each other.
• A cell with two overlapping nuclei were scored only if the nuclear boundaries of each nucleus were distinguishable.
• The cytoplasmic boundary or membrane of a binucleated cell was intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells.
- Criteria for scoring micronuclei (MNi): Micronuclei which had the following characteristics were considered for evaluation:
• The diameter of MNi in human lymphocytes usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei, which corresponds to 1/256th and 1/9th of the area of one of the main nuclei in a BN cell, respectively.
• Micronuclei (MNi) were non-refractile and therefore were readily distinguished from artifacts such as staining particles.
• Micronuclei (MNi) were not linked or connected to the main nuclei.
• Micronuclei (MNi) might touch but were not overlapping with the main nuclei and the micronuclear boundary was distinguishable from the nuclear boundary.
• Micronuclei (MNi) usually had the same staining intensity as the main nuclei but occasionally staining might be more intense.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): As no increase in Frequency of micronuclei was seen, characterization of micronuclei for whole or fragmented chromosomes by kinetochore antibody binding was not required/applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: replication index

Rationale for test conditions:

Test condiitions were those described by the relevant OECD test guideline

Evaluation criteria:

Assay considered negative if the following criteria were met:
- None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- There was no concentration-related increase when evaluated with an appropriate trend test (e.g., Kraskal-wallis).
- All results were inside the distribution of the historical control data.

Statistics:

Data on micronuclei containing binucleated cells were subjected to Shapiro-Wilk’s test for normality and Bartlett’s test to assess homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet suitable homogeneity of variance, Student's t-test was performed to determine the level of significance between Negative control, three selected test concentrations (selected based on the replicative index data) and positive controls.

Key result

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.24 - 7.35
- Data on osmolality: 296 - 316 mOsm/kg H2O
- Possibility of evaporation from medium: No
- Water solubility: Soluble in selected vehicle (water) at 200 µL/mL
- Precipitation and time of the determination: None observed
- Definition of acceptable cells for analysis: Frequency of viable mono-nucleated, bi- nucleated and multinucleated cells was measured to determine cytostatic effects and the rate of mitotic division. Cell types having the following characteristics were considered for evaluation:
• Mono-nucleated, bi- nucleated and multinucleated cells should be with an intact cytoplasm and normal nucleus morphology containing one, two and three or more nuclei, respectively.
• These cells may or may not contain one or more micronuclei.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
Preliminary cytotoxicity assay at 6 concentrations. Cytostasis observed in the absence of the metabolic activation was 1.87%, 2.29%, 4.46%, 6.30%, 10.84% and 18.65% at concentrations of 0.0625, 0.125, 0.25, 0.5, 1, and 2 µL/mL, respectively and in the presence of metabolic activation was 0.34%, 2.50%, 4.84%, 7.95%, 5.73% and 11.91% at 0.0625, 0.125, 0.25, 0.5, 1, and 2 µL/mL, respectively

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The number of binucleated cells with micronuclei in the negative control cultures were within the published range (Fenech, M., 2007: Cytokinesis-block micronucleus cytome assay, Nature Protocols, Vol. 2/5, pp. 1084-1104). The positive control, cyclophosphamide, produced a significant increase in the frequency of micronuclei containing binucleated cells (4.736% compared with a negative control value of 0.349%) following short term exposure in the presence of metabolic activation. The positive control, vinblastine, produced a significant increase (mean value of positive control was 3.977% MNBN against a negative control mean value of 0.342% MNBN negative control of 0.342) in the frequency of micronuclei containing binucleated cells following continuous exposure in the absence of metabolic activation.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : There was no concentration-related increase when evaluated with an appropriate trend test
- Statistical analysis; p-value if any : No statistically significant difference between treated cells and the negative control was apparent

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Without metabolic activation - range 0.29 - 5.57; mean 3.80; standard deviation 0.90; 95% control limits 0.00 - 7.70; number of data 36
With metabolic activation - range 2.37 - 6.74; mean 3.91; standard deviation 0.75; 95% control limits 0.00 - 7.86; number of data 36
- Negative (solvent/vehicle) historical control data:
Without metabolic activation - range 0.10 - 0.60; mean 0.36; standard deviation 0.14; 95% control limits 0.00 - 1.56; number of data 36
With metabolic activation - range 0.10 - 0.60; mean 0.35; standard deviation 0.13; 95% control limits 0.00 - 1.53; number of data 36

Summary of Cytostasis

 

Short-term treatment

Concentration	Absence of Metabolic Activation		Presence of Metabolic Activation
	% RI (Mean)	Cytostasis	% RI (Mean)	Cytostasis
NC (Distilled water) #	100	0	100	0
0.125 µL/mL	110.10	-10.10	102.54	-2.54
0.25 µL/mL	90.05	9.95	101.28	-1.28
0.5 µL/mL#	93.12	6.88	99.93	0.07
1 µL/mL#	92.62	7.38	109.58	-9.58
2 µL/mL #	104.03	-4.03	96.21	3.79
PC#	-	-	79.74	20.26

Key:  NC = Negative control, T = Treatment group, PC = Positive control (Cyclophosphamide @ 30 µg/mL in the presence of metabolic activation), # = Concentrations selected for scoring of binucleated cells containing micronuclei, RI = Replicative index.

Long-term treatment

Absence of Metabolic Activation
Concentration	% RI (Mean)	Cytostasis
NC (Distilled water) #	100	0
0.125 µL/mL	99.33	0.67
0.25 µL/mL	103.65	-3.65
0.5 µL/mL#	102.02	-2.02
1 µL/mL#	105.38	-5.38
2 µL/mL#	97.16	2.84
PC#	96.06	3.94

Key:  NC = Negative control, T = Treatment group, PC = Positive control (Vinblastine @ 0.008 µg/mL in the absence of metabolic activation), # = Concentrations selected for scoring of binucleated cells containing micronuclei, RI = Replicative index.

Individual Observation of Slides for Frequency of Micronucleated Binucleated Cells

Short-term treatment

Concentration	R	Absence of Metabolic Activation			Presence of Metabolic Activation
		Total number of BN cells scored	Number of MNBN	Percent MNBN	Total number of BN cells scored	Number of MNBN	% MNBN
NC (Distilled water)	1	1004	5	0.498	1005	5	0.498
	2	1002	3	0.299	1002	2	0.200
T3 (0.5 µL/mL)	1	1003	4	0.399	1003	4	0.399
	2	1007	2	0.199	1006	2	0.199
T4 (1 µL/mL)	1	1002	4	0.399	1005	3	0.299
	2	1003	3	0.299	1004	2	0.199
T5 (2 µL/mL)	1	1002	3	0.299	1004	4	0.398
	2	1001	5	0.500	1002	3	0.299
PC	1	NA			1002	47	4.691
	2				1004	48	4.781

 

Long-term treatment

Concentration	R	Total Number of BN Cells Scored	Number of MNBN	% MNBN
NC (Distilled water)	1	1020	4	0.392
	2	1029	3	0.292
T3 (0.5 µL/mL)	1	1000	4	0.400
	2	1002	2	0.200
T4 (1 µL/mL)	1	1004	5	0.498
	2	1102	3	0.272
T5 (2 µL/mL)	1	1002	4	0.399
	2	1005	2	0.199
PC	1	1007	30	2.979
	2	1005	50	4.975

Key: R = Replicate,T = Treatment group, NC = Negative control, BN cells = Binucleated cells, MNBN = Micronucleated binucleated cells, PC = Positive control (Cyclophosphamide 30 µg/mL in the presence of metabolic activation (short-term treatment) and Vinblastine 0.008 µg/mL in the absence of metabolic activation (long-term treatment)), NA = Not applicable.

Conclusions:

2-(2 -ethoxyethoxy) ethyl acetate does not induce micronuclei and does not have clastogenic or aneugenic potential in isolated human peripheral blood lymphocytes both in the absence and presence of a metabolic activation system.

Executive summary:

The clastogenic and aneugenic potential of EDGA 2 -(2 -ethoxyethoxy)ethyl acetate has been investigated in human peripheral blood lymphocytes that have undergone cell division after exposure to the substance in the absence and presence of metabolic activation. Methods used were in accordance with OECD test guideline 487.

The results from the study demonstrate that2 -(2 -ethoxyethoxy) ethyl acetate does not induce micronuclei and does not have clastogenic or aneugenic potential in isolated human peripheral blood lymphocytes both in the absence and presence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Available data regarding the potential genotoxicity of 2 -(2 -ethoxyethoxy)ethyl acetate (CAS 112 -15 -2) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and the data are therefore conclusive but not sufficient for classification.