2,2'-oxydiethanol

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:

27 Nov - 17 Dec 2012

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)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-oxydiethanol

EC Number:

203-872-2

EC Name:

2,2'-oxydiethanol

Cas Number:

111-46-6

Molecular formula:

C4H10O3

IUPAC Name:

2-(2-hydroxyethoxy)ethan-1-ol

Specific details on test material used for the study:

- Name of test material (as cited in study report): Diethylene glycol
- Physical state: colorless clear liquid
- Analytical purity: 99.8 % (BASF SE analytical report 12L00357)

Method

Target gene:

- His operon for S. typhimurium strains
- Trp operon for the E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: WP2: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix prepared from Wistar rats treated with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on three consecutive days.

Test concentrations with justification for top dose:

First experiment (standard plate test, with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: good solubility of the test item in the vehicle

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylendiamine (NOPD)

Details on test system and experimental conditions:

STANDARD PLATE TEST (SPT)
According to Ames et al., Mut Res 31: 347-364 (1975) and Maron & Ames, Mut Res 113: 173-215 (1983)

In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42 to 45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.

PREINCUBATION TEST (PIT)
According to Yahagi et al. Mut Res 48: 121-129 (1977) and Matsushima et al., In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens, Springer Verlag Berlin, Heidelberg, New York (1980)

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of either S9 mix or phosphate buffer were incubated at 37°C for 20 minutes. After addition of 2 mL soft agar, samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.

Evaluation criteria:

An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+8/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Results and discussion

Test results

Test results 1

Species / strain:

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

Remarks:

SPT

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

Species / strain:

E. coli WP2 uvr A

Remarks:

SPT

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 3

Species / strain:

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

Remarks:

PIT

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 4

Species / strain:

E. coli WP2 uvr A

Remarks:

PIT

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation was detected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxic effects were seen in either the standard plate test (SPT) or the preincubation test (PIT) with and without metabolic activation.

CONTROLS
Negative and positive controls were as expected and confirmed the validity and sensitivity of the test method and system.

Any other information on results incl. tables

Experiment 1: Standard plate-incorporation test

SPT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	14	48	8	21	103
33	10	51	8	15	100
100	12	49	9	19	103
333	12	53	7	20	106
1000	13	47	7	18	100
2500	15	52	9	21	104
5000	12	55	6	18	104
Respective positive control	1354	949	331	723	1048
SPT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	17	57	9	28	111
33	16	60	8	27	104
100	21	57	10	27	105
333	16	63	10	28	108
1000	16	62	11	32	112
2500	18	66	8	26	104
5000	17	61	9	29	113
Respective positive control	461	1225	664	776	196
Experiment 2: Preincubation test PIT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	10	47	6	34	98
33	12	46	9	31	97
100	12	42	8	34	101
333	10	48	9	32	107
1000	10	52	6	31	103
2500	10	52	7	31	99
5000	13	45	7	37	109
Respective positive control	1639	1345	868	890	986
PIT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	13	51	11	49	108
33	12	53	10	47	107
100	11	60	9	44	106
333	12	56	11	47	107
1000	11	48	13	47	108
2500	12	51	10	48	103
5000	12	58	13	46	105
Respective positive control	512	1440	313	1224	301