Butyl ethyl ether

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:

2016-11-08 to 2016-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

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:

Butyl ethyl ether

EC Number:

211-055-7

EC Name:

Butyl ethyl ether

Cas Number:

628-81-9

Molecular formula:

C6H14O

IUPAC Name:

1-ethoxybutane

Test material form:

liquid

Details on test material:

- Source and batch No.of test material: 60930023
- Expiration date of the batch: 09/2021
- Purity: 98.4%
- Physical State: liquid
- Density: 0.75 g/cm³
- Colour: colourless
- Storage condition of test material: Keep container tightly closed. Store in a dry and cool area

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible
with the survival of the bacteria and the S9 activity.

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Source of cells: MOLTOX, INC., NC 28607, USA (TA 98, 1535 and 102), Xenometrix AG, Switzerland (TA 100 and 1537).
Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre
Selective Agar: 2% Vogel-Bonner-Glucose-Minimal Agar
Overlay Agar: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-hisitidne x HCl x H2O, 12.2 mg Biotin per litre

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (Conditions: see "Any other information on materials and methods"; Results: see "Any other information on results" Table 2). 5.0 μL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Controls 3

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 4

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation (experiment I)); preincubation (experiment II)
- Cell density at seeding (if applicable): approx. 10^9 cells/mL 100µL/plate

EXPERIMENTAL PROCEDURE
- Experiment I:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method 100 μL of the test item preparation was pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h at least

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 2.5 µL/plate without S9 and at 5 µL/plate with S9 in TA 98, TA 100, TA 1535 and TA 1537. At 5 µL/plate without S9 in TA 102

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I. In experiment II in tester strains TA 98, TA 100, TA 1535 and TA 1537 toxic effects of the test item were noted at concentrations of 2.5 µL/plate and higher (without metabolic activation) and at 5.0 µL/plate with metabolic activation). In tester strain TA 102 toxic effects of the test item were noted at a concentration of 5.0 µL/plate (without metabolic activation). No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with butyl ethyl ether at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Remarks on result:

other: Experiment I (plate incorporation)

Any other information on results incl. tables

Results of the pre-experiment

Table 2: Results pre-experiment

		Dose (µL/plate)	TA 98		TA 100
			Mutation Factor [toxicity]*		Mutation Factor [toxicity]*
			without S9	with S9	without S9	with S9
Solvent Control (DMSO)			1.0	1.0	1.0	1.0
4-NOPD		10 µg	19.8	-	-	-
NaN3		10 µg	-	-	10.3	-
2-AA		2.5 µg	-	95.2	-	28.5
Test Item Butyl ethyl ether		0.00316	1.2	0.8	0.9	0.9
		0.0100	1.2	0.9	1.0	0.9
		0.0316	1.1	0.8	1.0	0.9
		0.100	1.4	1.0	1.1	0.9
		0.316	1.2	0.7	1.0	1.0
		1.0	0.9	0.8	1.0	1.1
		2.5	1.0	0.9	1.0	1.1
		5.0	1.2	0.9	1.0	1.1

* [toxicity parameter]: B = Background lawn reduced, N = No background lawn

Applicant's summary and conclusion

Conclusions:

Based on the results of this guideline study (OECD 471), butyl ethyl ether is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of S. typhimurium were exposed to butyl ethyl ether (98.4 % purity) in DMSO at concentrations of 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate in two individual experiments (plate-incorporation and pre-incubation) each in the presence and absence of mammalian metabolic activation. No cytotoxic effects were observed in experiment I, while in experiment II cytotoxicity was seen in TA 98, TA 100, TA 1535 and TA 1537 at concentrations of 2.5 µL/plate and higher (without metabolic activation) and at the limit concentration (5.0 µL/plate) with metabolic activation. In tester strain TA 102 toxic effects were noted at a concentration of 5.0 µL/plate (without metabolic activation). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments. Therefore, butyl ethyl ether is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation assay).