2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990-11-27 to 1990-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

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:

2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

EC Number:

237-655-9

EC Name:

2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

Cas Number:

13887-98-4

Molecular formula:

C8H14O7

IUPAC Name:

2-{2-[2-(carboxymethoxy)ethoxy]ethoxy}acetic acid

Test material form:

other: liquid: clear colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D 269
- Expiration date of the lot/batch: stable until December 1991
- Purity test date: 1990-07-16

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dark at approximately 5 °C
- Stability under test conditions: stable until December 1991, stability in solvent proved for 4 hours

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

The test compound was tested at doses of 4 to 10000 microgram/plate and proved to be toxic to the bacterial strains at doses of 2500 microgram/ plate. Thinning of the bacterial lawn and a reduction in the number of colonies have been observed at this dose.
Concentrations: 0, 4, 20, 100, 500, 2500, 5000, 10000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: soluble in water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant colonies were counted


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

standard test conditions for this assay

Evaluation criteria:

A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.

Statistics:

Statistical analysis is not neccessary as only the number of colonies has to be compared to the controls.

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:

cytotoxicity

Remarks:

cytotoxicity observed at doses of 2500 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

Cytotoxicity was observed in the preliminary experiment at doses of 2500 µg/plate and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

cytotoxicity observed at doses of 2500 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

cytotoxicity observed at doses of 2500 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity observed at doses of 2500 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

	strain TA 100		strain TA 1535		Strain TA 1537		strain TA 1538		strain TA 98
conc. [µg] per plate	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	171	169	10	9	5	5	12	14	19	28
4	158	161	8	9	4	4	13	19	14	27
20	163	170	9	11	5	7	13	10	19	26
100	158	166	10	9	6	8	15	14	18	23
500	158	163	7	9	6	10	11	10	20	24
2500	140	144	9	12	8	6	14	15	15	25
10000	123	153	8	9	12	6	8	12	21	25
Sodium azide 1 µg	558		434
9-Aminoacridine 50 µg					282
2-Nitrofluorene 2.5 µg							1234		1019
2-Aminoanthracene 0.5 µg (TA 100, TA1538, TA98) 1µg (TA1535, TA1537)		725		178		124		474		484
Benzo[a]pyrene 10 µg		1316		35		128		202		588

 

 

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

	strain TA 100		strain TA 1535		Strain TA 1537		strain TA 1538		strain TA 98
conc. [µg]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	111	124	9	9	8	10	16	19	18	25
4	105	124	6	11	11	13	13	21	20	27
20	99	124	8	8	8	9	12	24	25	25
100	116	124	10	9	9	7	14	20	22	28
500	115	124	10	8	7	12	10	22	21	22
2500	113	105	6	12	7	6	11	25	28	18
10000	105	100	7	8	7	7	17	20	24	23
Sodium azide 1 µg	494		375
9-Aminoacridine 50 µg					246
2-Nitrofluorene 2.5 µg							874		652
2-Aminoanthracene 0.5 µg (TA 100, TA1538, TA98) 1µg (TA1535, TA1537)		570		130		121		323		347
Benzo[a]pyrene 10 µg		1198		18		126		131		616

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate (S9 mix, Aroclor 1254 induced). A dose range of 6 different doses from 4 microgram/plate to 10000 microgram/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test compound proved to be toxic to the bacterial strains at 2500 microgram/plate. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test item did not result in relevant increases in the number of revertant colonies. Summarizing, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.