Butanedioic acid, 2-sulfo-, 1(or 4)-[2-(C12-18 and C18-unsatd. amido)ethyl] esters, disodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods, therefore the study is considered relevant, adequate and reliable for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

IUPAC Name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

Test material form:

other: liquid

Method

Target gene:

histidine

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:

S9

Test concentrations with justification for top dose:

Main tests: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
In a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test, ten concentrations (0.316, 1.0, 3.16. 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg act. ingr. per plate) were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.
Hence, 5000 µg act. ingr./plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injection (aqua ad iniectabilia)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide in aqua ad iniectabilia

Remarks:

(10 µg/plate) TA100 and TA1535 without S9

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene in DMSO

Remarks:

(10 µg/plate) TA98 without S9

Controls 3

Negative solvent / vehicle controls:

yes

Positive control substance:

other: 9-amino-acridine in ethanol, abs.

Remarks:

(100 µg/plate) TA 1537 without S9

Controls 4

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Mitomycin C in DMSO

Remarks:

(10 µg/plate) TA 102 without S9

Controls 5

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Benzo(a)pyrene in DMSO

Remarks:

(10 µg/plate) TA 98, TA 102 and TA 1537 with S9

Controls 6

Negative solvent / vehicle controls:

yes

Positive control substance:

other: 2-amino-anthracene in DMSO

Remarks:

(2 µg/plate) TA 100, TA 1535 with S9

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min (2nd experiment)
- Exposure duration: 48-72 h (1st and 2nd experiment)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate (3 plates/concentration and strain)

DETERMINATION OF CYTOTOXICITY
- Method: other: a reduction in the number of spontaneous revertants, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures

Evaluation criteria:

The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
- at one or more concentrations the number of revertants is reproducibly increased in at least one strain with or without metabolic activation. A 2-fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA 98, TA 100 and TA 102. For the strains TA 1535 and TA 1537 a 3-fold increase represents a biological relevant effect. The Mann and Whitney test (p ≤ 0.05) may be used to determine statistical significance.
Or
- a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.
- Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Statistics:

The Mann and Whitney test (p ≤ 0.05) may be used to determine statistical significance.
The Spearman's rank correlation coefficient may be applied.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]¬esters, disodium salts tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test substance was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle served as the negative control.

Preliminary test

The test substance was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations ranging from 0.316 to 5000 µg act.ingr./plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.

Hence, 5000 µg act.ingr./plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 3.16 to 5000 µg act.ingr./plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg act.ingr./plate in all test strains.

No increase in revertant colony numbers as compared with control counts was observed up to a concentration of 5000 µg act.ingr./plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions the test substance tested up to a concentration of 5000 µg act.ingr./plate, caused no mutagenic effect in theSalmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.