Sodium 2-butoxyethyl sulphate

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:

5 Oct 2017 - 7 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

Országos Gyógyszerészeti és Élelmezés-egészégügyi Intézet

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-butoxyethyl sulphate

EC Number:

266-840-7

EC Name:

Sodium 2-butoxyethyl sulphate

Cas Number:

67656-24-0

Molecular formula:

C6H14O5S.Na

IUPAC Name:

sodium 2-butoxyethyl sulphate

Test material form:

solid

Method

Target gene:

his, trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Rathenau Str. 2, D-35394 Giessen, Germany
- Methods for maintenance in cell culture if applicable: The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions of cultures on nutrient agar plates.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: ready-to-use minimal glucose agar (MGA) plates
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically 'cleansed' against high spontaneous background: not reported

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Trinova Biochem GmbH, Rathenau Str. 2, D-35394 Giessen, Germany
- Methods for maintenance in cell culture if applicable:
The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions of cultures on nutrient agar plates.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
ready-to-use minimal glucose agar (MGA) plates
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically 'cleansed' against high spontaneous background: not reported

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50 or 16 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water (ASTM Type 1) and dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: In the preliminary Solubility and Concentration Range Finding Tests ultrapure water (ASTM Type 1) was found as appropriate vehicle for preparing the test item solutions. This vehicle is compatible with the survival of the bacteria and the S9 activity. DMSO also was used depending on the solubility of the test item and the solubility of positive control reference items.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD, 4 μg/plate, -S9, TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test for the initial mutation test and a pre-incubation test for the confirmatory mutation test

DURATION
- Preincubation period: 11-13 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Conditions were investigated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development

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 vehicle 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 vehicle control.

A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically.

The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate 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 titers are in the 109 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 each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

Statistics:

Not performed.

Results and discussion

Test results

Test results 1

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:

not examined

Positive controls validity:

valid

Test results 2

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:

not examined

Positive controls validity:

valid

Test results 3

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:

not examined

Positive controls validity:

valid

Test results 4

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:

not examined

Positive controls validity:

valid

Test results 5

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 examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary of the Initial Mutation Test

Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	TA98				TA100				TA1535				TA1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate and mutation rate(MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
UntreatedControl	26.3	0.99	30.0	1.23	99.3	1.07	105.3	0.90	13.7	1.11	11.3	0.97	9.7	0.97	11.3	1.06	47.0	1.09	54.7	1.12
DMSOControl	23.7	1.00	21.0	1.00	–	–	94.0	1.00	–	–	9.0	1.00	6.3	1.00	11.3	1.00	–	–	48.3	1.00
UltrapureWaterControl	26.7	1.00	24.3	1.00	92.7	1.00	117.3	1.00	12.3	1.00	11.7	1.00	10.0	1.00	10.7	1.00	43.0	1.00	49.0	1.00
5000	30.3	1.14	29.0	1.19	89.0	0.96	106.3	0.91	11.7	0.95	10.3	0.89	8.0	0.80	11.0	1.03	53.0	1.23	56.7	1.16
1600	29.0	1.09	27.0	1.11	93.0	1.00	105.0	0.89	12.0	0.97	9.7	0.83	8.3	0.83	8.0	0.75	47.3	1.10	56.7	1.16
500	21.0	0.79	26.3	1.08	86.7	0.94	113.0	0.96	11.7	0.95	9.7	0.83	8.0	0.80	9.7	0.91	43.3	1.01	55.3	1.13
160	20.0	0.75	24.0	0.99	99.7	1.08	104.3	0.89	9.7	0.78	13.3	1.14	8.7	0.87	12.0	1.13	46.3	1.08	49.3	1.01
50	21.7	0.81	24.0	0.99	95.7	1.03	122.0	1.04	12.7	1.03	9.3	0.80	9.0	0.90	10.0	0.94	48.7	1.13	55.7	1.14
16	27.7	1.04	28.0	1.15	91.7	0.99	111.3	0.95	11.0	0.89	9.3	0.80	10.0	1.00	9.7	0.91	43.7	1.02	53.7	1.10
NPD (4µg)	323.3	13.66	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2µg)	–	–	–	–	1744.0	18.82	–	–	1458.7	118.27	–	–	–	–	–	–	–	–	–	–
9AA (50µg)	–	–	–	–	–	–	–	–	–	–	–	–	622.7	98.32	–	–	–	–	–	–
MMS (2µL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1085.3	25.24	–	–
2AA (2µg)	–	–	2666.7	126.98	–	–	3162.7	33.65	–	–	178.0	19.78	–	–	183.3	16.18	–	–	–	–
2AA(50µg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	241.0	4.99

Applicant's summary and conclusion

Conclusions:

No mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains without and with metabolic activation. The test substance is non-mutagenic in test strains used.