2-(2-ethoxyethoxy)-2-methylpropane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay. Under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation. The test item showed no biologically relevant increase of mutants in the in vitro mammalian cell gene mutation test after treatment with the test item (with and without metabolic activation).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-08-29 to 2013-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

5 mg/plate or 5 μL/plate were generally selected as maximum test dose.
1st experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
2nd experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Standard Plate Test and Preincubation Test both with and without metabolic activation
METHOD OF APPLICATION: Standard plate test based on the method of Ames et al. + Preincubation test based on the method described by Yahagi et al. and Matsushima et al.

Conditions
- Preincubation period: 20 minutes using a shaker
- Exposure duration: 48 - 72 hours
- Temperatur: Incubation at 37 °C

Titer determination
- The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer was recorded for all test groups both with and without S9 mix in all experiments and indicated
in the tables.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system. A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Standard plate test					Preincubation test
	TA1535							TA1535
Dose (µg)	Without S9 Mix		With S9 Mix	Dose (µg)		Without S9 Mix			With S9 Mix
DMSO	11		11	DMSO		11			11
33	13		12	33		10			11
100	11		11	100		11			13
333	12		11	333		10			12
1000	12		11	1000		12			9
2500	11		10	2500		11			10
5000	12		11	5000		10			11
MNNG	1130			MNNG		1198
2-AA			362	2-AA					453
	TA100					TA100
Dose (µg)	Without S9 Mix		With S9 Mix	Dose (µg)		Without S9 Mix			With S9 Mix
DMSO	48		55	DMSO		59			63
33	48		52	33		59			67
100	49		47	100		56			65
333	51		52	333		58			64
1000	50		50	1000		59			63
2500	52		48	2500		61			64
5000	50		50	5000		59			62
MNNG	1201			MNNG		1477
2-AA			1339	2-AA					1606
	TA1537					TA1537
Dose (µg)	Without S9 Mix		With S9 Mix	Dose (µg)		Without S9 Mix			With S9 Mix
DMSO	5		7	DMSO		6			7
33	6		6	33		6			7
100	7		7	100		6			7
333	6		5	333		6			6
1000	6		6	1000		6			7
2500	6		7	2500		6			6
5000	5		8	5000		7			7
AAC	559			AAC		547
2-AA			234	2-AA					229
	TA98					TA98
Dose (µg)	Without S9 Mix		With S9 Mix	Dose (µg)		Without S9 Mix			With S9 Mix
DMSO	18		22	DMSO		16			18
33	20		24	33		16			19
100	17		24	100		14			17
333	19		27	333		15			19
1000	16		28	1000		16			17
2500	15		21	2500		15			19
5000	20		22	5000		15			17
NOPD	510			NOPD		864
2-AA			1239	2-AA					1153
	E.coli					E.coli
Dose (µg)	Without S9 Mix		With S9 Mix	Dose (µg)		Without S9 Mix			With S9 Mix
DMSO	74		63	DMSO		34			32
33	66		65	33		34			29
100	65		66	100		35			32
333	64		70	333		34			33
1000	80		68	1000		34			31
2500	65		67	2500		36			30
5000	69		66	5000		35			29
4-NQO	824			4-NQO		1278
2-AA			554	2-AA					434

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames-Test

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD guideline 471, EU method B.13/14 and EPA OPPTS 870.5100. The dose range tested was 33 µg/plate - 5000 µg/plate (Standard plate test and preincubation test) with and without metabolic activation. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

In vitro Micronucleus Test

The substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 187.50; 375.00; 750.00; 1500.00 μg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In this study, no clear cytotoxicity indicated by reduced cell counts or proliferation index (CBPI) was observed up to the highest required test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

In vitro Mammalian Cell Gene Mutation Test (HPRT Locus)

The test item was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The selection of the concentrations was based on data from the pre-experiment. Experiment I and II with and without metabolic activation were performed as a 4 h short-term exposure assay. The test item was investigated in duplicate cultures at the following concentrations:

Experiment I

with and without metabolic activation:

0.5, 1.0, 2.5, 5.0, 7.5, 10.0 mM (1460 μg/mL)

Experiment II

with and without metabolic activation:

0.75, 2.0, 4.0, 6.0, 8.0, 10.0 mM (1460 μg/mL)

No precipitation of the test item was noted in the experiments. Biologically relevant growth inhibition (reduction of relative growth below 70%) was observed in experiment I and II without metabolic activation. In experiment I without metabolic activation the relative growth was 44. 1 % ( culture A) and 47.1% (culture B) for the highest concentration (10.0 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 10.0 mM with a relative growth of 87.0% (culture A) and 80.9% (culture B). In experiment II without metabolic activation the relative growth was 69.9% (culture A) and 64.3% ( culture B) for the highest concentration (10.0 mM) evaluated. The highest concentration evaluated with metabolic activation was 10.0 mM with a relative growth of72.2% (culture A) and 70.1% (culture B). In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation No 605/2014.