Butanedioic acid, sulfo-, C-[2-[(1-oxododecyl)amino]ethyl] ester, monosodium salt

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:

Apr. - July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-{2-[(Z)-(1-oxidododecylidene)amino]ethoxy}-4-oxo-2-sulfobutanoate 4-{2-[(Z)-(1-oxidododecylidene)amino]ethoxy}-4-oxo-3-sulfobutanoate

EC Number:

939-648-2

Cas Number:

75081-73-1

Molecular formula:

C18H31NNa2O8S

IUPAC Name:

tetrasodium 4-{2-[(Z)-(1-oxidododecylidene)amino]ethoxy}-4-oxo-2-sulfobutanoate 4-{2-[(Z)-(1-oxidododecylidene)amino]ethoxy}-4-oxo-3-sulfobutanoate

Test material form:

other: Coarse white pieces

Method

Target gene:

Histidine (his- → his+)
Tryptophane (trp- → trp+)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: TA98, TA1535 and E. coli are obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA100 and TA1537 are obtained from Xenometrix AG, Switzerland. Stored as stock cultures with nutrient broth (OXOID)/DMSO (approx. 8% v/v) over liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 :
The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gießen, Germany.

- method of preparation of S9 mix:
Male Wistar rats are induced with phenobarbital (80 mg/kg bw) and β- naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins) or male Sprague Dawley rats are induced with phenobarbital/ β-naphthoflavone (Trinova).
A stock of the supernatant containing the microsomes is frozen in aliquots between 2 and 5 mL and stored at
- concentration of S9 mix:
The protein concentration in the S9 preparation of Eurofins Munich was 34.4 mg/mL (Lot: 070521), and in the S9 preparation of Trinova, 37.8 mg/mL (Lot: 4423). The protein concentration of Lot No.: 4423 was adjusted to 30 mg/mL

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
at Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test

at Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and mutation induction with each three plates:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate.
The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
As no precipitation nor limiting toxicity of the test item was observed in either tester strain used in the pre-experiment, 5.0 μL/plate was selected as the maximum concentration to be used in experiment I.

Vehicle / solvent:

- Justification for choice of solvent/vehicle:
solubility

The test item was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 2.55 was applied to consider the active components of the test item.

Controls

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD, 4-nitro-o-phenylene-diamine, 10 µg/plate; 2-AA, 2-aminoanthracene, 2.5 µg/plate (S. thyphimurium) or 10 µg/plate (E. coli)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate
- Number of independent experiments : 2, if first experiment negative or equivocal

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Samples of each tester strain are grown by culturing for 12 h at 37 °C in S. typhimurium medium (Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 109 cells/mL).
- Test substance added:
1. experiment - in agar (plate incorporation)
2. experiment - preincubation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 60 min.
- Incubation after exposure: 48 h at 37°C on selective medium

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

according to current OECD guideline

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In tester strain E. coli WP2 uvrA (pKM101), toxic effects of the test item were noted at concentrations of 2.5 μL/plate and higher (without metabolic activation).

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

please refer to attachment

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 μL/plate
(TA98, TA100, TA1535, TA1537)
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
(E. coli WP2 uvrA (pKM101))

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
The microbial contamination observed in one plate (experiment II, TA98, 0.0316 μL/plate, with metabolic activation) did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella typhimurium revertants and thus did not affect the evaluation.
Toxic effects of the test item were observed in experiment I in tester strains TA98 and TA100 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strain TA1535, toxic effects of the test item were noted at concentrations of 1.0 μL/plate and higher (without metabolic activation) and at a concentration of 5.0 μL/plate (with metabolic activation). In tester strain TA1537 toxic effects of the test item were observed at a concentration of 5.0 μL/plate (without metabolic activation).
In experiment II, toxic effects of the test item were noted in tester strains TA98, TA100, TA1535 and TA1537 at concentrations of 0.316 μL/plate and higher (with and without metabolic activation). In tester strain E. coli WP2 uvrA (pKM101), toxic effects of the test item were noted at concentrations of 2.5 μL/plate and higher (without metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.