2-(oxolan-2-ylmethoxy)ethanol

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:

12 July - 21 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in Aug 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(oxolan-2-yl)methoxy]ethan-1-ol

EC Number:

608-659-1

Cas Number:

31692-85-0

Molecular formula:

C5H10O2[C2H4O]n, n = 0, 1, 2, 3, 4, ... (data given for max n of 4)

IUPAC Name:

2-[(oxolan-2-yl)methoxy]ethan-1-ol

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:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Prior to the main experiments, a range-finding study was performed using the TA 98 and TA 100 strains with following test concentrations: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 μL/plate with and without metabolic activation.
The following six concentrations were then used in the two main experiments: 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 μL/plate with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent: good compatibility with the survival of the bacteria and the S9 activity

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first main experiment); preincubation (second confirmatory experiment)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and inspection of the bacterial background lawn, toxicity is determined by a clearing or diminution of the background lawn or reduction of the number of revertants to a mutation factor of approximatively ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

Acceptance criteria
The study was considered valid if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative/solvent control plates with and without S9 mix are within the historical control data ranges
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

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.

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.

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 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: There was no reduced background lawn and barely any change in the mutation factor of the treated cells (TA98: -S9 Mix: 0.9 - 1.3, +S9 Mix: 0.9 - 1.2; TA100: -S9 Mix: 0.9 - 1.2, +S9 Mix: 1.0 - 1.2) compared to the solvent control. The positive controls induced a reversion rate more than two times higher than the reversion rate of the solvent control.

HISTORICAL CONTROL DATA (2014 - 2016)
- Positive historical control data:
Range values revertants, without S9:
TA 98 (4-NOPD): 141 - 1830
TA 100 (SA): 132 - 1423
TA 1535 (SA): 38 - 1854
TA 1537 (4-NOPD): 35 - 273
TA 102 (MMS): 272 - 3321
Range values revertants, with S9:
TA 98 (2AA): 70 - 3606
TA 100 (2AA): 169 - 3132
TA 1535 (2AA): 22 - 1954
TA 1537 (2AA): 26 - 682
TA 102 (2AA): 137 - 3588

- Negative (solvent/vehicle) historical control data:
Range values revertants, without S9:
TA 98: 11 - 58
TA 100: 49 - 155
TA 1535: 4 - 41
TA 1537: 3 - 35
TA 102: 141 - 472
Range values revertants, with S9:
TA 98: 15 - 59
TA 100: 62 - 160
TA 1535: 3 - 38
TA 1537: 3 - 36
TA 102: 157 - 586

Any other information on results incl. tables

Table 1:Summary of test results (experiment 1; Initial Mutation Test, Plate Incorporation Method)

With or without S9-Mix	Test substance concentration (μL/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	TA102
–	Solvent control (distilled water)	22 ± 2.1	29 ± 5.0	81 ± 13.6	21 ± 3.5	194 ± 29.4
	0.0316	20 ± 1.5	26 ± 7.6	84 ± 3.1	25 ± 3.6	204 ± 12.2
	0.100	21 ± 1.5	33 ± 4.2	76 ± 7.0	22 ± 3.0	170 ± 1.5
	0.316	21 ± 1.5	31 ± 1.7	99 ± 11.7	25 ± 3.0	164 ± 13.3
	1.0	21 ± 3.1	29 ± 3.0	78 ± 8.3	25 ± 5.5	199 ± 15.0
	2.5	22 ± 3.2	32 ± 7.9	78 ± 6.1	22 ± 5.7	206 ± 19.7
	5.0	20 ± 1.2	31 ± 10.6	98 ± 15.0	26 ± 5.0	158 ± 14.0
	Positive controls (unit/plate)	4-NOPD (40 µg)	4-NOPD (10 µg)	SA (10 µg)	SA (10 µg)	MMS (1 µL)
	Mean No. of colonies/plate (average of 3 plates)	170 ± 13.3	480 ± 136.3	372 ± 43.1	751 ± 42.4	1042 ± 227.1
+	Solvent control (distilled water)	21 ± 0.6	25 ± 7.5	67 ± 0.6	31 ± 4.6	281 ± 5.9
	0.0316	19 ± 3.1	22 ± 0.0	73 ± 9.0	30 ± 5.0	279 ± 13.2
	0.100	20 ± 2.5	27 ± 4.4	68 ± 12.9	24 ± 5.6	273 ± 8.5
	0.316	18 ± 1.7	30 ± 0.6	78 ± 11.8	30 ± 5.3	281 ± 27.6
	1.0	19 ± 3.2	23 ± 3.5	79 ± 12.5	26 ± 5.6	264 ± 12.4
	2.5	18 ± 1.2	26 ± 9.5	66 ± 15.1	31 ± 2.1	275 ± 31.8
	5.0	19 ± 3.2	28 ± 3.1	73 ± 4.6	25 ± 5.0	291 ± 12.1
	Positive controls (µg/plate)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (10)
	Mean No. of colonies/plate (average of 3 plates)	413 ± 41.9	1797 ± 236.1	872 ± 113.3	175 ± 25.1	894 ± 58.4

SD = standard deviation

NPD = 4-nitro-1,2-phenylene-diamine

SA = sodium azide

MMS = methylmethanesulfonate

2AA = 2-aminoanthracene

Table 2: Summary of test results (experiment 2; Confirmatory Mutation Test, Pre-Incubation Method)

With or without S9-Mix	Test substance (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	TA102
–	Solvent control (ultrapure water)	8 ± 3.5	34 ± 13.1	92 ± 10.0	15 ± 2.0	209 ± 6.5
	0.0316	9 ± 0.6	31 ± 2.6	86 ± 8.0	22 ± 4.0	222 ± 36.0
	0.100	8 ± 3.1	25 ± 1.5	101 ± 10.4	17 ± 0.6	235 ± 13.8
	0.316	12 ± 1.5	32 ± 4.2	89 ± 12.7	22 ± 5.8	211 ± 3.5
	1.0	7 ± 3.2	33 ± 7.0	98 ± 6.1	19 ± 3.1	212 ± 11.1
	2.5	9 ± 3.6	29 ± 6.4	88 ± 16.0	21 ± 7.6	193 ± 64.6
	5.0	12 ± 1.7	29 ± 1.5	92 ± 8.4	18 ± 3.8	194 ± 13.8
	Positive controls (unit/plate)	4-NOPD (40 µg)	4-NOPD (10 µg)	SA (10 µg)	SA (10 µg)	MMS (1 µL)
	Mean No. of colonies/plate (average of 3 plates)	89 ± 7.5	365 ± 25.7	368 ± 36.3	1146 ± 41.9	1462 ± 357.9
+	Solvent control (DMSO)	14 ± 2.5	28 ± 9.0	72 ± 18.0	15 ± 5.0	234 ± 22.5
	0.0316	15 ± 1.7	31 ± 3.8	74 ± 0.6	11 ± 2.5	245 ± 14.3
	0.100	11 ± 2.1	34 ± 7.5	91 ± 2.9	17 ± 2.6	247 ± 23.4
	0.316	15 ± 3.5	31 ± 1.2	71 ± 12.1	12 ± 1.5	251 ± 17.0
	1.0	12 ± 3.0	33 ± 8.1	83 ± 18.9	13 ± 2.9	266 ± 10.5
	2.5	13 ± 1.7	36 ± 4.0	77 ± 15.9	15 ± 3.8	274 ± 31.8
	5.0	12 ± 3.2	36 ± 4.7	80 ± 7.0	14 ± 1.5	272 ± 44.2
	Positive controls (µg/plate)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (10)
	Mean No. of colonies/plate (average of 3 plates)	128 ± 10.7	1470 ± 191.8	976 ± 229.7	134 ± 15.0	690 ± 38.2

SD = standard deviation

NPD = 4-nitro-1,2-phenylene-diamine

SA = sodium azide

MMS = methylmethanesulfonate

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.