trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

EC Number:

948-778-9

Molecular formula:

C14O8H19N2Na3

IUPAC Name:

trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

Test material form:

liquid

Details on test material:

Active ingredient (%): 48.9% dry substance
Purity (%): 95.50 %
Stability: 5 years
Sterilization: None
Solubility: Soluble in water
Storage: Room Temperature

Method

Target gene:

The phenotypic characteristics checked, were: Histidine requirement, presence or absence of R-factor plasmids where appropriate (i.e. Ampicillin resistance in strains TA 98, TA 100 and Ampicillin + Tetracycline resistance in strain TA 102); the presence of characteristic mutations as UVrB and rfa mutation.

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:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

The test sample has been tested at:
1) 5 mg/plate
2) 1.5 mg/plate
3) 0.5 mg/plate
4) 0.15 mg/plate
5) 0.05 mg/plate

Vehicle / solvent:

The test item at 50 mg/ml in water and 4 subsequent dilutions in water of semi-log intervals between them have been prepared.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

mitomycin C

other: Daunomycin

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments :2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): n/a
- Test substance added : in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
Microbiological controls
Aliquots of maximum concentration of the assay samples, Solvent, PBS, S9 mix and Top Agar, were directly placed in TSA and MGA plates. The development of any bacterial colonies have been observed after 48-72 hours of incubation at 37°C±1°C and recorded.
- Exposure duration/duration of treatment: 37±1°C for 48-72 hours.
- Harvest time after the end of treatment (sampling/recovery times): none

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition;
The sample toxicity assay for the test strains was determining any reduction in culture growth and number
of spontaneously reverting organisms caused by the assay sample.

Evaluation criteria:

ASSAY VALIDITY CRITERIA
In the microbiological controls performed on MGA and TSA plates, top-agar, solvent used, assay sample at the highest concentration tested, PBS and S9 Mix should not be contaminated by more than two colonies per plates.
On average, for the TA 1535, TA 1537 and TA 98 strains, the number of reverting colonies developed by the positive control should be at least 300% higher than that of the respective negative control (lower limit for positive control/negative control ratio for strains with low mutation frequency); for the TA100 and TA102 strains, the number of reverting colonies developed by the positive control should be at least 200% higher than that of the respective negative control (a 20% of variation is accepted at the lower limit of the positive control/negative control ratio in strains with high mutation frequency).
These criteria refer to internal method validation only. OECD 471 guideline doesn't give any reference thereof.
The average number of spontaneously reverting colonies per plate in the negative controls should be included between the validated limits.

Results and discussion

Test results

Test results 1

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

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 3

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 4

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 5

Species / strain:

S. typhimurium TA 102

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

Remarks on result:

other: not mutagenic

Any other information on results incl. tables

Assay validity criteria

Microbiological controls:the microbiological controls performed on the assay sample at maximum concentration prepared, on Top Agar supplemented, solvents, PBS and S9mix, didn't show any contamination.

Negative and positive controls: the average number of spontaneously reverting colonies in the negative control plates did not exceed the established limits and all positive controls caused a significant increase of number of reverting colonies.

Genetic characteristics of the bacterial strains: the verification of the genetic characteristics showed that the test strains maintained the required genetic properties in both the assay repetitions.

Toxicity of the test substance

On the basis of the results obtained during the test, the test substance did not show toxic effects either in

the presence or absence of the enzymatic system for metabolism activation.

Mutacienic Activity

Neither reproducible increase in the number of revertants colonies per plate in any strain with or without metabolic activation system was detected. Besides, the statistical test applied showed no significant difference between the numbers of revertants colonies for assay sample vs. negative control.

Applicant's summary and conclusion

Conclusions:

On the basis of the results interpreted according to OECD 471:1997, the test substance "TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE" tested as recommended by Standards, proved to be NOT MUTAGENIC for all the test strains, either in the presence or absence of metabolic activation.

Executive summary:

On the test item "TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE", a genotoxicological study was carried out to evaluate its mutagenic effects. The following test was performed:

- Bacterial reverse mutation assay (Ames test).

The Bacterial reverse mutation assay was performed on five mutant strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, TA 102).

The presumed mutagenic activity of the test substance was determined by comparing the number of reverting colonies in treated cultures with the number of the reverting organisms in the control cultures. The direct incorporation method in a plate was used both in the presence of, and without, an enzymatic system for metabolic activation.

The test item has been tested at 50 mg/mL, and 4 subsequent dilutions of semi-log intervals between them.

The assay was performed in two replicates.

On the basis of the results interpreted according to OECD 471:1997, the test substance "TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE" tested as recommended by Standards, proved to be NOT MUTAGENIC for all the test strains, either in the presence or absence of metabolic activation.