Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts

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:

29 March 2017 to 07 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 4-sec-decyl, ammonium salts

Molecular formula:

C16H29NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-decyl, ammonium salts

Constituent 2

Reference substance name:

Benzenesulfonic acid, 4-sec-undecyl, ammonium salts

Molecular formula:

C17H31NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-undecyl, ammonium salts

Constituent 3

Reference substance name:

Benzenesulfonic acid, 4-sec-dodecyl, ammonium salts

Molecular formula:

C18H33NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-dodecyl, ammonium salts

Constituent 4

Reference substance name:

Benzenesulfonic acid, 4-sec-tridecyl, ammonium salts

Molecular formula:

C19H35NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-tridecyl, ammonium salts

Test material form:

other: Highly viscous light yellow liquid

Details on test material:

Storage Conditions: Highly viscous light yellow liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stable at room temperature
- Stability under test conditions: Not required
- Solubility and stability of the test substance in the solvent/vehicle: Not required
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not required

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article stock solutions were prepared by the test article under subdued lighting in purified water with the aid of vortex mixing, ultrasonication and warming at 37 °C, to give the maximum required treatment concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm/CR filter, 0.2 µm pore size) and subsequent dilutions were made using purified water. The test article solutions were protected from light and used within approximately 2 hours of initial formulation.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 50, 16, 5, 1.6, 0.5, 0.16 and 0.05 mg/mL treatment solutions prepared.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as an aqueous formulation.

OTHER SPECIFICS: n/a

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Experiment 1 (S9 ±): 5, 16, 50, 160, 500, 1600, 5000 µg/plate
Experiment 2 (S9 -): 0.5, 1.6, 5, 16, 50, 160, 500 µg/plate
Experimetn 2 (S9 +): 15, 30, 60, 125, 250, 500, 1000 µg/plate

A maximum concentration of 5000 µg/plate was selected for Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). For Experiment 2 the maximum concentration tested was selected on the basis of toxicity seen in the assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Test item is completely soluble in water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Mutation Experiment

Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45 ± 1 °C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
This was followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37 ± 1 °C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section 4.4).
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step in order to increase the sensitivity of the test system. Quantities of test article, vehicle control solution or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37 ± 1 °C, with shaking, before the addition of 2 mL molten agar at 45 ± 1 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

Toxicity Assessment

The background lawns of the plates were examined for thinning as sign of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

Statistical analysis using Dunnett’s test was used to aid evaluation of a concentration response, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

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:

Toxicity observed at 1600 µg/plate. Slightly thinned bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate. Slight thinning of bacterial lawn at 160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slight thinning of bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate. Slight thinning of bacterial lawn observed at 160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slight thinning of bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate. Slight thinning of the bacterial lawn observed at 160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 1600 µg/plate. Slight thinning of bacterial lawn observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity observed at 5000 µg/plate. Very thin bacterial lawn present at 1600 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: test item readily soluble at highest tested concentration
- Precipitation: not obsered
- Definition of acceptable cells for analysis: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).
- Other confounding effects: n/a

RANGE-FINDING/SCREENING STUDIES: no

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: visual inspection of bacterial lawn
- Other observations when applicable: n/a

Any other information on results incl. tables

Table 2:       Mean number of revertants per plate (experiment 1 – pre-incubation not conducted)

Conc. (µg/plate)	TA98			TA100			TA1535			TA1537			TA102
	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)
Neg. control	27.3	49.3	- / -	124.3	89.3	- / -	18.7	15.3	- / -	12.7	8.0	- / -	260.0	353.3	- / -
5	21.7	33.0	- / -	104.7	91.7	- / -	12.7	16.0	- / -	12.3	13.0	- / -	279.0	344.0	- / -
16	25.7	29.3	- / -	95.7	107.3	- / -	19.7	12.0	- / -	7.5	14.0	- / -	252.3	316.7	- / -
50	29.3	39.0	- / -	94.0	109.3	- / -	16.3	12.3	- / -	10.0	14.3	- / -	254.3	362.7	- / -
160	22.7	22.7	- / -	62.3	95.3	S / -	18.0	20.3	S / -	8.7	9.3	S / -	185.7	422.0	- / -
500	9.0	25.0	S / S	0	80.0	T / S	0	15.0	T / S	0	12.7	T / S	0	342.0	T / -
1600	0	0	T / T	0	0	T / T	0	0	T / T	0	0	T / T	0	116.0	T / V
5000	0	0	T / T	0	0	T / T	0	0	T / T	0	0	T / T	0	0	T / T
Pos. control	545.3	102.0	- / -	528.0	785.3	- / -	631.7	224.0	- / -	321.7	426.3	- / -	904.3	3078.3	- / -

T= cytotoxic, no sign of revertant colonies

- = no sign of cytotoxicity

S= slight reduction in bacterial lawn

V= very thin bacterial lawn

^*= p<0.05

^**= p<0.01

 

Table 3:       Mean number of revertants per plate (experiment 2 – pre-incubation conducted)

Conc. (µg/plate)	TA98		TA100		TA1535		TA1537		TA102
	S9 -	Toxicity	S9 -	Toxicity	S9 -	Toxicity	S9 -	Toxicity	S9 -	Toxicity
Neg. control	22.7	-	100.0	-	13.3	-	10.0	-	259.7	-
0.5	25.7	-	90.0	-	14.7	-	8.0	-	325.3	-
1.6	23.7	-	78.0	-	11.7	-	14.7	-	299.0	-
5	25.7	-	83.0	-	11.3	-	9.3	-	305.7	-
16	27.7	-	98.7	-	11.0	-	6.0	-	294.0	-
50	30.3	-	90.3	-	21.0	-	7.7	-	260.7	-
160	28.0	-	75.3	-	12.3	-	6.3	-	201.0	-
500	22.3	-	13.0	V	10.7	-	1.3	-	126.7	S
Pos. control	591.0	-	885.3	-	642.0	-	227.7	-	722.0	-

 

Conc. (µg/plate)	TA98		TA100		TA1535		TA1537		TA102
	S9 +	Toxicity	S9 +	Toxicity	S9 +	Toxicity	S9 +	Toxicity	S9 +	Toxicity
Neg. control	33.3	-	101.0	-	18.0	-	17.7	-	298.3	-
15	45.7^*	-	109.7	-	15.0	-	16.0	-	372.3^*	-
30	33.7	-	117.0	-	13.0	-	11.0	-	390.3^**	-
60	36.7	-	88.3	-	14.3	-	10.3	-	340.7	-
125	31.0	-	91.3	-	13.7	-	9.3	-	360.0	-
250	39.7	-	82.3	-	15.0	-	11.0	-	349.7	-
500	29.7	-	96.7	-	17.7	-	8.7	-	244.7	-
1000	19.0	-	24.7	-	14.3	-	7.3	-	218.7	-
Pos. control	303.0	-	1296.3	-	177.3	-	427.0	-	1552.0	-

- = no sign of cytotoxicity

S= slight reduction in bacterial lawn

V= very thin background bacterial lawn

T= cytotoxic, no revertant colonies

^*= p<0.05

^**= p<0.01

Applicant's summary and conclusion

Conclusions:

It was concluded that under the condition of this study, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines).

Executive summary:

OECD 471 (2017) - In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were exposed to Benzenesulfonic acid, 4 -C10 -13 -sec-alkyl derivs., ammonium salts, formulated in purified water at concentration range of 0.5 – 5000 µg/plate in the presence and absence of mammalian metabolic activation S9-mix.

The test item was tested up to 5000 µg/plate (or its cytotoxic limit, where relevant), the maximum recommended concentration according to OECD 471.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence (or a concentration related positive response) of induced mutant colonies over background in the test item treated plates.

This study is classified acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.