Sodium (2-butoxyethoxy)acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames test) according to OECD TG 471, the test item did not induce gene mutations in the tester strains used.

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:

March 21, 2018 - April 5, 2018

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:

21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) reversion system measures his- to his+ reversions.
The E. coli strain WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC) resulting in trp- to trp+ reversions.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Selection of the concentrations for the initial mutation test is based on the results of the solubility test and the concentration range finding test (informatory toxicity test).
The test item concentrations for the initial and confirmatory mutation tests were as follows:
5000, 1600, 500, 160, 50 and 16 μg/plate (with and without S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: The test item is soluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine, NPD

Remarks:

without S9 mix, TA98, 4 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA100 and TA1535, 2 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, TA1537, 50 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 mix, E.coli, 2 µL/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

with S9 mix, TA98,TA100,TA1535,TA1537 (each 2 µg/plate); E.coli (50 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37ºC in a shaking incubator
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number

Evaluation criteria:

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 for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control
Criteria for a negative response:
A test article is considered non-mutagenic 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:

None

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: yes, clear solution up to 50 mg/mL
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES
In the informatory toxicity test the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.
In the informatory toxicity test a cytotoxic effect of the test item on the bacterial strains was not observed. The colony and background lawn development was not affected in any case. All of the observed slight revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system. The obtained revertant colony numbers were sporadically higher than the revertant colony numbers of the solvent control; however all of the observed higher colony numbers remained within the biological variability range of the applied test system and far below the biologically relevant threshold for being positive.
Based on the results of the preliminary test, the maximum test item concentration was 5000 µg/plate (±S9 mix).

HISTORICAL CONTROL DATA
- Positive historical control data: see table 3
- Negative (solvent/vehicle) historical control data: see table 4

Table 1: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	15.0	0.83	20.7	0.89	79.0	1.05	99.7	0.87	9.7	1.26	7.0	0.91	5.7	1.31	9.0	1.08	32.0	0.91	33.3	0.76
DMSO Control	12.0	1.00	22.0	1.00	–	–	114.7	1.00	–	–	8.7	1.00	6.0	1.00	6.0	1.00	–	–	41.7	1.00
Ultrapure Water Control	18.0	1.00	23.3	1.00	75.0	1.00	115.0	1.00	7.7	1.00	7.7	1.00	4.3	1.00	8.3	1.00	35.0	1.00	44.0	1.00
5000	19.3	1.07	17.7	0.76	89.0	1.19	99.7	0.87	12.7	1.65	8.7	1.13	4.7	1.08	7.0	0.84	36.3	1.04	52.7	1.20
1600	14.3	0.80	17.3	0.74	86.0	1.15	101.0	0.88	8.3	1.09	11.0	1.43	4.0	0.92	7.3	0.88	29.0	0.83	47.3	1.08
500	15.7	0.87	18.7	0.80	86.0	1.15	97.3	0.85	9.0	1.17	7.3	0.96	6.7	1.54	6.3	0.76	32.3	0.92	51.0	1.16
160	17.3	0.96	27.0	1.16	82.7	1.10	96.0	0.83	8.0	1.04	8.7	1.13	6.0	1.38	7.0	0.84	34.7	0.99	38.0	0.86
50	16.7	0.93	19.3	0.83	82.7	1.10	94.3	0.82	6.3	0.83	8.3	1.09	5.7	1.31	8.0	0.96	28.7	0.82	38.7	0.88
16	20.0	1.11	19.0	0.81	89.3	1.19	95.0	0.83	7.3	0.96	11.3	1.48	4.3	1.00	9.7	1.16	32.0	0.91	54.0	1.23
NPD (4 µg)	396.7	33.06	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 µg)	–	–	–	–	786.7	10.49	–	–	1282.7	167.30	–	–	–	–	–	–	–	–	–	–
9AA (50 µg)	–	–	–	–	–	–	–	–	–	–	–	–	320.7	53.44	–	–	–	–	–	–
MMS (2 µL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	768.7	21.96	–	–
2AA (2µg)	–	–	2005.3	91.15	–	–	1893.3	16.51	–	–	223.7	25.81	–	–	85.0	14.17	–	–	–	–
2AA (50µg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	217.0	5.21

MR: Mutation Rate;         NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA:9-Aminoacridine; MMS:Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:           Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	14.7	0.64	18.3	0.93	74.3	0.89	93.0	1.09	10.7	1.07	14.0	1.11	7.0	1.17	6.7	1.05	23.0	0.86	27.3	0.80
DMSO Control	15.0	1.00	20.3	1.00	–	–	77.3	1.00	–	–	11.3	1.00	7.3	1.00	7.3	1.00	–	–	23.7	1.00
Ultrapure Water Control	23.0	1.00	19.7	1.00	83.3	1.00	85.7	1.00	10.0	1.00	12.7	1.00	6.0	1.00	6.3	1.00	26.7	1.00	34.0	1.00
5000	17.0	0.74	17.3	0.88	74.0	0.89	79.0	0.92	12.3	1.23	12.0	0.95	7.3	1.22	8.0	1.26	33.7	1.26	25.3	0.75
1600	16.0	0.70	24.0	1.22	81.3	0.98	85.3	1.00	10.3	1.03	12.7	1.00	7.3	1.22	6.0	0.95	32.3	1.21	26.0	0.76
500	18.0	0.78	25.3	1.29	83.7	1.00	85.3	1.00	8.0	0.80	12.7	1.00	5.7	0.94	7.7	1.21	36.0	1.35	30.3	0.89
160	14.7	0.64	22.0	1.12	78.0	0.94	96.3	1.12	11.7	1.17	11.3	0.89	7.7	1.28	8.0	1.26	28.7	1.08	32.0	0.94
50	11.7	0.51	22.0	1.12	85.7	1.03	98.0	1.14	12.7	1.27	12.0	0.95	7.7	1.28	8.7	1.37	30.3	1.14	25.7	0.75
16	19.7	0.86	24.0	1.22	74.7	0.90	87.0	1.02	9.3	0.93	10.3	0.82	7.3	1.22	7.0	1.11	30.3	1.14	24.7	0.73
NPD (4 µg)	493.3	32.89	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 µg)	–	–	–	–	828.7	9.94	–	–	1013.3	101.33	–	–	–	–	–	–	–	–	–	–
9AA (50 µg)	–	–	–	–	–	–	–	–	–	–	–	–	549.3	74.91	–	–	–	–	–	–
MMS (2 µL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	993.3	37.25	–	–
2AA (2 µg)	–	–	1150.3	56.57	–	–	830.7	10.74	–	–	192.3	16.97	–	–	117.7	16.05	–	–	–	–
2AA (50 µg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	174.0	7.35

MR: Mutation Rate;         NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA:9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:             Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 3: Historical Control Values of positive control for Revertants/Plate (for the Period of 2015-2017)

			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	285.9	1214.0	1024.3	594.4	858.4
		SD	25.5	80.0	120.3	36.4	164.7
		Minimum	152	609	407	136	330
		Maximum	598	2272	2597	2048	1760
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1395.7	1727.4	160.7	145.8	204.9
		SD	261.4	244.1	30.0	18.9	3.9
		Minimum	286	712	91	70	133
		Maximum	3211	3435	328	315	367

Table 3: Historical Confor Revertants/Plate for the Period of 2015-2017)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	19.7	94.6	10.9	8.9	25.2
		SD	1.7	2.4	0.4	1.3	5.2
		Minimum	8	67	4	3	11
		Maximum	40	133	21	20	52
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	24.5	112.9	11.0	9.2	31.7
		SD	1.4	6.1	0.5	1.2	6.4
		Minimum	11	74	3	3	13
		Maximum	43	159	20	20	60
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	18.1	87.0	10.8	8.5	24.6
		SD	1.1	3.6	0.6	1.3	3.3
		Minimum	9	58	4	3	10
		Maximum	36	131	23	20	54
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	23.0	102.4	11.0	9.2	31.4
		SD	0.8	7.7	0.4	1.3	5.8
		Minimum	11	69	3	3	12
		Maximum	42	148	23	21	59
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	19.5	92.8	11.3	9.1	26.6
		SD	1.4	5.0	0.5	1.8	6.6
		Minimum	12	62	4	3	10
		Maximum	30	139	22	18	52
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	24.4	109.9	11.2	9.5	34.0
		SD	1.2	6.7	0.8	1.9	6.1
		Minimum	13	83	5	4	16
		Maximum	37	149	18	18	63

Conclusions:

The test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

A study according OECD TG 471 was performed to investigate the potential mutagenic activity of the test item using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility test and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I).

At the preparation of the test item stock solution any correction factor (based on its purity, constituents) was not taken into consideration.

Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50 and 16 µg/plate.

The results of the preliminary concentration range finding test allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with sodium (2-butoxyethoxy) acetate at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study according OECD TG 471 was performed to investigate the potential mutagenic activity of the test item using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility test and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I).

At the preparation of the test item stock solution any correction factor (based on its purity, constituents) was not taken into consideration.

Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50 and 16 µg/plate.

The results of the preliminary concentration range finding test allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with sodium (2-butoxyethoxy) acetate at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test itemdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information on the test item regarding genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.