2,2'-[ethylenebis(oxy)]bisacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the bacterial reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-10-02 to 1990-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D 271

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 / 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.
In the second experiment, 5000 µg/plate was chosen as the highest dose.

Vehicle / solvent:

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

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

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

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

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

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.

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

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

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

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

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

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	162	178	8	8	12	15	23	22	28	34
4	168	180	9	17	15	16	16	18	26	33
20	176	180	11	9	12	16	20	18	34	34
100	196	198	11	11	15	14	18	18	29	34
500	201	204	10	8	13	14	16	20	25	29
2500	168	177	13	13	11	17	17	19	27	35
10000	147	134	5	10	12	10	17	17	33	25
Sodium azide 1 µg	639		472
9-Aminoacridine 50 µg					133
2-Nitrofluorene 2.5 µg							597		527
2-Aminoanthracene 0.5 µg (TA 100, TA1538, TA98) 1µg (TA1535, TA1537)		992		191		133		820		855
Benzo[a]pyrene 10 µg		1661		32		109		229		724

 

 

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	141	153	12	13	7	8	16	18	21	23
4	154	155	12	11	5	6	14	15	25	25
20	140	153	11	9	7	9	19	16	24	29
100	162	164	9	16	7	9	14	17	21	34
500	147	149	8	11	9	8	16	16	23	27
2500	161	145	8	8	7	7	17	15	25	32
10000	134	152	13	12	8	7	17	11	25	28
Sodium azide 1 µg	705		555
9-Aminoacridine 50 µg					621
2-Nitrofluorene 2.5 µg							1332		835
2-Aminoanthracene 0.5 µg (TA 100, TA1538, TA98) 1µg (TA1535, TA1537)		1094		184		115		973		1161
Benzo[a]pyrene 10 µg		1396		32		118		197		604

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

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.

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 for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.