2-ethoxyethyl ethyl carbonic acid ester

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:

08 February 2013 to 23 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxyethyl ethyl carbonic acid ester

EC Number:

809-934-0

Cas Number:

627034-93-9

Molecular formula:

C7H14O4

IUPAC Name:

2-ethoxyethyl ethyl carbonic acid ester

Test material form:

liquid

Details on test material:

- Appearance: transparent colourless liquid
- Storage conditions: Refrigerated (2-8 °C), under inert gas, protected from light

Method

Target gene:

Salmonella typhimurium
TA1537: his C 3076; rfa-; uvrB- (frame shift mutations)
TA98: his D 3052; rfa-; uvrB-; R-factor (frame shift mutations)
TA1535: his G 46; rfa-; uvrB- (base-pair mutations)
TA100: his G 46; rfa-; uvrB-; R-factor (base-pair mutations)

Escherichia coli
WP2 uvrA: trpE; uvrA- (base-pair substitution)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Range Finding Test: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate

Initial Mutation Test and Confirmatory Mutation Test: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the same concentrations were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test material was examined using distilled water, dimethyl sulfoxide (DMSO) and acetone. The test material was soluble in acetone and DMSO at 100 mg/mL concentration, partial dissolution was observed in distilled water at the same concentration. Due to the better biocompatibility to the test system, DMSO was selected for vehicle of the study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO; distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; 4-nitro-1,2-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
A standard plate incorporation procedure was performed, as an Initial Mutation Test.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes was as follows: top agar 2000 μL, solvent or test material solution (or reference controls) 50 μL, overnight culture of test strain 100 μL, phosphate buffer (pH 7.4) or S9 mix 500 μL.
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no clear positive effect was observed. Before the overlaying, the test material formulation (or vehicle/solvent or reference control), the bacterial culture, and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test material (in its vehicle/solvent).
The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS: 3

EVALUATION OF EXPERIMENTAL DATA
The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using Microsoft Excel™ software.

VALIDITY CRITERIA
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analysable concentrations were presented in all strains of the main tests.

Evaluation criteria:

A test material was considered mutagenic if:
- a dose-related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the spontaneous reversion rate of the negative (solvent) control plates.

A test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and 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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Preliminary Range Finding Test, the numbers of revertant colonies were mostly in the normal range. Minor differences were detected in some cases, but they were without biological significant, thus they were considered as biological variability of the test system.
No insolubility or signs of cytotoxicity was observed in the preliminary experiment.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test using the plate incorporation method, the highest revertant rate was observed in the in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at the concentration of 1581 μg/plate. The mutation factor value was 2.40, slightly above the respective biological threshold value of 2. Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain. However, there was no dose response, and the numbers of revertant colonies were within the historical control range. Additionally, higher numbers of revertant colonies compared to the DMSO solvent control were detected for untreated control (MF: 1.47) and distilled water control (MF: 1.20) in this strain.

In the Confirmatory Mutation Test using the pre-incubation method, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain with metabolic activation at 500 μg/plate concentration. The observed mutation factor value was 1.48. However, no dose-dependent relationship was observed, and the observed mutation factor value was below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range.
Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some other cases. However, no dose-dependence was observed in any cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
The sporadic increases in the number of revertant colonies were examined for consistency; none of them were repeatable when comparing the performed main experiments. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between test material treated samples and negative (solvent) controls.
Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
No insolubility or signs of cytotoxicity was observed in the main tests.

VALIDITY OF THE TESTS
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests.
The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Any other information on results incl. tables

Table 1: Summary Table of Results of the Initial Mutation Test

Concentrations (µg/plate)	Mean values of revertants/ Mutation factor (MF)	Tester strain
		TA98		TA100				TA1535		TA1537		WP2 uvrA
		-S9	+S9		-S9	+S9	-S9		+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	23.0	35.0		91.0	119.7	7.3		10.0	12.7	11.0	35.3	37.7
	MF	0.99	1.02		0.99	1.09	1.47		0.94	1.09	1.00	0.88	1.47
DMSO control	Mean	23.3	34.3		91.7	109.3	5.0		10.7	11.7	11.0	40.0	25.7
	MF	1.00	1.00		1.00	1.00	1.00		1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	─	─		97.7	─	6.0		─	─	─	47.0	─
	MF	─	─		1.07	─	1.20		─	─	─	1.18	─
5000	Mean	29.7	26.3		94.7	121.3	8.0		11.7	10.3	8.7	40.0	45.3
	MF	1.27	0.77		1.03	1.11	1.60		1.09	0.89	0.79	1.00	1.77
1581	Mean	29.3	29.0		88.0	129.0	12.0		9.3	13.0	15.7	53.0	45.3
	MF	1.26	0.84		0.96	1.18	2.40		0.88	1.11	1.42	1.33	1.77
500	Mean	28.3	28.0		97.7	109.3	9.7		8.3	12.3	12.3	47.0	46.3
	MF	1.21	0.82		1.07	1.00	1.93		0.78	1.06	1.12	1.18	1.81
158.1	Mean	28.7	31.3		91.3	123.0	8.0		11.3	12.3	11.0	48.3	44.3
	MF	1.23	0.91		1.00	1.13	1.60		1.06	1.06	1.00	1.21	1.73
50	Mean	28.7	27.0		99.0	109.7	6.7		11.3	16.3	9.7	52.0	47.7
	MF	1.23	0.79		1.08	1.00	1.33		1.06	1.40	0.88	1.30	1.86
15.81	Mean	27.0	27.0		101.7	115.0	9.0		10.7	11.3	12.0	33.3	48.7
	MF	1.16	0.79		1.11	1.05	1.80		1.00	0.97	1.09	0.83	1.90
5	Mean	25.7	31.3		103.0	116.7	9.0		7.7	9.0	12.0	39.7	47.7
	MF	1.10	0.91		1.12	1.07	1.80		0.72	0.77	1.09	0.99	1.86
NPD (4µg)	Mean	320.0	─		─	─	─		─	─	─	─	─
	MF	13.71	─		─	─	─		─	─	─	─	─
2AA (2 µg)	Mean	─	2263.0		─	2828.0	─		250.7	─	253.7	─	─
	MF	─	65.91		─	25.87	─		23.50	─	23.06	─	─
2AA (50 µg)	Mean	─	─		─	─	─		─	─	─	─	293.0
	MF	─	─		─	─	─		─	─	─	─	11.42
SAZ (2 µg)	Mean	─	─		1700.7	─	656.3		─	─	─	─	─
	MF	─	─		17.41	─	109.39		─	─	─	─	─
9AA (50 µg)	Mean	─	─		─	─	─		─	380.0	─	─	─
	MF	─	─		─	─	─		─	32.57	─	─	─
MMS (2 µL)	Mean	─	─		─	─	─		─	─	─	988.3	─
	MF	─	─		─	─	─		─	─	─	21.03	─

 

Table 2: Summary Table of Results of the Confirmatory Mutation Test

Concentrations (µg/plate)	Mean values of revertants/ Mutation factor (MF)	Tester strain
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	34.3	40.0	94.3	124.0	7.7	14.7	7.7	8.7	36.7	44.7
	MF	0.99	1.15	0.95	1.08	0.61	1.29	0.82	0.96	0.99	0.92
DMSO control	Mean	34.7	34.7	99.7	115.3	12.7	11.3	9.3	9.0	37.0	48.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	─	─	105.0	─	9.0	─	─	─	43.7	─
	MF	─	─	1.05	─	0.71	─	─	─	1.18	─
5000	Mean	32.7	44.0	108.3	118.3	8.3	11.0	9.7	7.7	36.3	46.3
	MF	0.94	1.27	1.09	1.03	0.66	0.97	1.04	0.85	0.98	0.96
1581	Mean	33.7	37.0	96.0	112.0	6.7	9.0	10.0	6.0	37.0	45.0
	MF	0.97	1.07	0.96	0.97	0.53	0.79	1.07	0.67	1.00	0.93
500	Mean	33.7	37.3	105.3	107.7	7.0	10.7	6.7	13.3	38.0	45.0
	MF	0.97	1.08	1.06	0.93	0.55	0.94	0.71	1.48	1.03	0.93
158.1	Mean	32.3	38.7	96.0	116.0	5.3	5.3	5.7	7.3	39.0	48.0
	MF	0.93	1.12	0.96	1.01	0.42	0.47	0.61	0.81	1.05	0.99
50	Mean	31.7	32.3	110.7	119.7	7.7	7.0	7.0	7.0	33.0	45.0
	MF	0.91	0.93	1.11	1.04	0.61	0.62	0.75	0.78	0.89	0.93
15.81	Mean	32.0	33.7	91.3	117.7	9.3	8.7	6.3	8.3	35.0	43.3
	MF	0.92	0.97	0.92	1.02	0.74	0.76	0.68	0.93	0.95	0.90
5	Mean	27.7	34.0	100.7	118.3	8.0	11.7	6.3	7.0	31.7	43.0
	MF	0.80	0.98	1.01	1.03	0.63	1.03	0.68	0.78	0.86	0.89
NPD (4µg)	Mean	296.0	─	─	─	─	─	─	─	─	─
	MF	8.54	─	─	─	─	─	─	─	─	─
2AA (2 µg)	Mean	─	2263.3	─	2501.3	─	212.7	─	215.3	─	─
	MF	─	65.29	─	21.69	─	18.76	─	23.93	─	─
2AA (50 µg)	Mean	─	─	─	─	─	─	─	─	─	274.7
	MF	─	─	─	─	─	─	─	─	─	5.68
SAZ (2 µg)	Mean	─	─	1292.7	─	1117.3	─	─	─	─	─
	MF	─	─	12.31	─	124.15	─	─	─	─	─
9AA (50 µg)	Mean	─	─	─	─	─	─	457.3	─	─	─
	MF	─	─	─	─	─	─	49.00	─	─	─
MMS (2 µL)	Mean	─	─	─	─	─	─	─	─	1187.3	─
	MF	─	─	─	─	─	─	─	─	27.19	─

Applicant's summary and conclusion

Conclusions:

The test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, test material had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, EPA OPPTS 870.5100, under GLP conditions. The method followed also conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

The study was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test, an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Solubility Test, the test material was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, most of the observed revertant colony numbers were below the respective biological threshold value. The observed sporadic increases in the number of revertant colonies were examined for consistency; none of them were repeatable when comparing the performed main experiments. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between test material treated samples and solvent controls; Thus, they were considered as biological variability of the test system.

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The findings of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test material had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.