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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in the Ames test.

Link to relevant study records

Reference

Reference 1

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

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 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

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

Vehicle / solvent:

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

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	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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-07-18 to 2017-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

only one bacterial strain tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

only one bacterial strain tested

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: 03/2016_R_JM
- Expiration date of the lot/batch: 26.06.2018
- Purity test date: 03.02.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25 °C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

OTHER SPECIFICS:
The test item used in this assay with a purity of 80.8 % consists of 11 % water as a constituent. The substance that is to be registered (dried test item with only 1 % water left) has a purity of 92.3 %. Drying process of the substance is only limited technically feasible. Therefore, at the preparation of test item stock solutions (for Initial and Confirmatory Mutation Tests) a correction factor of 1.14 (92.3/80.8=1.14) was applied in order to take the test item purity into account.

Target gene:

trpE,
in addition to tryptophan mutation, the Escherichia coli WP2 uvrA strain has additional mutations which enhance its sensitivity to mutagens. The uvrA strains are defective in excision repair.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: bacterial disc cultures from Trinova Biochem GmbH (Rathenau Str. 2),
35394 Giessen, Germany
- Cell cycle length, doubling time or proliferation index:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates: VWR International, Merck Life Science GmbH, Germany,
Tryptophan overlay agar (Agar Bacteriological, NaCl, Nutrient Broth, L-Tryptophan)

Additional strain / cell type characteristics:

other: defective in excision repair

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

16, 50, 160, 500, 1600, 5000 µg/plate
The toxicity of the test item was determined with Escherichia coli WP2 uvrA in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water was applied as vehicle of the test item and the positive control substance MMS

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water was applied as vehicle of the test item and the positive control substance MMS and DMSO was applied as vehicle for positive control substance 2AA.

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) (experiment 1) and preincubation test (experiment 2)

DURATION
- Exposure duration: 20 min
- Expression time (cells in growth medium): appr. 48 h

SELECTION AGENT (mutation assays): agar plates containing trypthophan

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all colonies were counted


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

according to guideline

Evaluation criteria:

The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- The Escherichia WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10^9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at the tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- clearing or diminution of the background lawn (reduced background lawn development occurs).

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response (the number of reversions is at least three times higher than the reversion rate of the vehicle control) for at least one of the dose groups occurs in the examined strain with or without metabolic activation.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The colony numbers on the controls (untreated, vehicle, positive) and the test item plates were determined (counted manually, evaluated by unaided eye), the mean values and appropriate standard deviations and mutation rates were calculated.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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(plate incorporation test)

Initial Mutation Test (Plate Incorporation Test)
Concentrations (μg/plate)			Escherichia coliWP2uvrA
-S9			+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR		Mean	MR
Untreated Control	31.3	0.84		43.3	1.10
DMSO Control	–	–		32.3	1.00
Ultrapure Water Control	37.3	1.00		39.3	1.00
5000	46.3	1.24		45.0	1.14
1600	36.0	0.96		51.0	1.30
500	41.7	1.12		47.0	1.19
160	40.7	1.09		52.0	1.32
50	39.7	1.06		44.3	1.13
16	34.0	0.91		51.0	1.30
MMS (2μL)	765.3	20.50		–	–
2AA (50 μg)	–	–		223.0	6.90

 

 

Table 2: Number of revertants per plate (mean of three plates), Experiment 2(Pre-Incubation Test)

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (μg/plate)			Escherichia coliWP2uvrA
-S9			+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR		Mean	MR
Untreated Control	26.7	1.03		35.0	0.95
DMSO Control	–	–		35.0	1.00
Ultrapure Water Control	26.0	1.00		36.7	1.00
5000	31.3	1.21		34.3	0.94
1600	27.3	1.05		39.7	1.08
500	34.7	1.33		33.7	0.92
160	33.0	1.27		35.3	0.96
50	26.0	1.00		35.3	0.96
16	31.3	1.21		38.3	1.05
MMS (2μL)	1168.0	44.92		–	–
2AA (50 μg)	–	–		273.0	7.80

 

Conclusions:

The test item is not mutagenic in Escherichia coli WP2 uvrA.

Executive summary:

The Escherichia coli WP2 uvrA tester strain was used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix, Phenobarbital (PB) and β-naphthoflavone (BNF) induced). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. No substantial increases were observed in revertant colony numbers of the investigated Escherichia coli WP2 uvrA tester strain following treatment at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strain at any examined concentration level (±S9 Mix) throughout the study. As a conclusion the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test (Salmonella typhimurium strains)

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.

 

Ames test (Echerichia coli WP2 uvrA)

The Escherichia coli WP2 uvrA tester strain was used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (± rat liver S9 Mix, Phenobarbital (PB) and β-naphthoflavone (BNF) induced). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. No substantial increases were observed in revertant colony numbers of the investigated Escherichia coli WP2 uvrA tester strain following treatment at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strain at any examined concentration level (±S9 Mix) throughout the study. As a conclusion the test item is considered non-mutagenic in this bacterial reverse mutation assay.

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.