calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 21st to May 05th, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Constituent 1

Reference substance name:

Set Retard Plus

IUPAC Name:

Set Retard Plus

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Tester strains of Salmonella typhimurium containing a different type of mutation in the histidine operon were employed for this study.

The strains are tested routinely for other genotypic characters such as:
- Histidine requirement – The His - character of all tester strains was confirmed by demonstrating the histidine requirement for growth on selective agar plates
- rfa mutation, i.e. cell membrane permeability was checked through sensitivity to crystalviolet
- uvrB mutation was checked through sensitivity to ultra-violet light (TA1535, TA98, TA100and TA97a)
- R- factor plasmid (pKM101)
- presence or absence of R- factor plasmid was checked through ampicillin resistance (TA98, TA100 and TA97a), and-pAQ1 plasmid
- the pAQ1 strain (TA102) was tested for both ampicillin and tetracycline resistance (TA102).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Test concentrations with justification for top dose:

5000, 1500, 500, 150 and 50 µg/plate

Vehicle / solvent:

- Vehicle: distilled water.

Controls

Untreated negative controls:

yes

Remarks:

Plates containing the reaction mixture, but not the test article formulation, and overlay agar supplemented with histidine, served as a negative control to check spontaneous revertants.

Negative solvent / vehicle controls:

yes

Remarks:

Since distilled water was the vehicle employed, plates containing the reaction mixture comprising of distilled water but not test item, and overlay agar supplemented with histidine, served as a control to check spontaneous revertants.

Positive controls:

yes

Remarks:

with and without S9 Mix

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-Aminoanthracene, 2- Aminofluorene, Danthron, ICR 191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).

DURATION
- Preincubation period: 12 to 16 hs at 37 °C to achieve cell density of about 1 x 10^9 cells/ml.
- Exposure duration: 48 hs.

NUMBER OF REPLICATIONS: triplicate plating was performed at each dose level.

DETERMINATION OF CYTOTOXICITY IN A PRELIMINARY TEST ON STRAIN TA100: relative total growth, in order to select test concentrations for main study.

PREPARATION OF MAMMALIAN LIVER FRACTION (S9)
Preparation of S9, using liver of Sprague Dawley rats and phenobarbitone with beta-naphthoflavone as inducing agents was carried out at INTOX laboratories following the method of Westphal et.al (2000).The S9 fraction was tested for protein content as well as sterility before preserving in cryocans at about -196 °C. For every experiment, fresh vials were removed from the cryocan, thawed and used in the preparation of S9 mix.

PREPARATION OF S9 MIX
The S9 mix was prepared in cold condition at ~ 4 °C, immediately prior to its use in the experiment.
The microsomal enzyme reaction mixture contained following components for 10 ml.
1) Rat liver S9 fraction1.00 ml
2) 0.4 M MgCl2, 1.65 M - KCl salt solution 0.20 ml
3) 1.0 M G-6-P0.05 ml
4) 0.1 M NADP0.40 ml
5) 0.2 M Phosphate buffer pH 7.4 5.00 ml
6) Distilled water3.35 ml

Evaluation criteria:

The mutagenic activity of the test article was assessed by applying the following criteria:
1. a test article is considered as mutagenic if it produces a concentration related increase over the range tested and /or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.The biologically significant increase would be assumed if the average colony counts are: at least twice as compared to those in vehicle control group for strain TA97a, TA98, TA100 and TA102 and at least thrice as compared to those in vehicle control group for strain TA1535.
2.a test article is considered as non-mutagenic, if it does not induce any increase in the number of revertants and does not show any dose response relationship, in two separate experiments, with any bacterial strain, either with or without metabolic activation.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA1535, TA97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

REVERTANT FREQUENCIES IN TREATMENT GROUPS
Test item was not found to be cytotoxic to strain TA100 as was observed during the preliminary cytotoxicity study. Frequencies of histidine revertant colonies observed at all concentrations of test item in tester strains TA1535, TA97a, TA98, TA100 and TA102, with and without the presence of a metabolic activation system, were comparable to those observed in the vehicle control groups, as per the criteria employed for evaluation of mutagenic potential, and this observation was confirmed by repetition of the experiments.

REVERTANT FREQUENCIES IN VEHICLE CONTROL GROUPS
Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at testing laboratory. They also compared well with the range reported in the published literature.

REVERTANT FREQUENCIES IN POSITIVE CONTROL GROUPS
Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:

Test item is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102, with and without metabolic activation.

Executive summary:

Method

Salmonella typhimurium Reverse Mutation Assay was carried out in compliance with the OECD Guidelines for Testing of Chemicals (No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997 and as per mutually agreed protocol. The test item was evaluated in the / Salmonella Plate Incorporation Assay to determine its ability to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9). Based upon the preliminary tests conducted to assess the solubility / precipitation and cytotoxicity of test item, the tester strains were exposed to the test article in triplicate cultures at the doses of 5000, 1500, 500, 150 and 50 µg/plate, both with and without metabolic activation system (S9). Liver S9, induced in Sprague Dawley rats by phenobarbitone with β-naphthoflavone, was used for this purpose. Distilled water was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine. The plates were incubated at 37 °C for 48 hours after which the histidine revertant colonies were counted and their frequency was compared with that in the vehicle control group. Concurrent negative control group and positive control groups were also included in the experiment, as specified by the test guideline.

Observations

Results of this test indicated that the frequencies of histidine revertant colonies at all concentrations of test item in strains TA1535, TA97a, TA98, TA100 and TA102, with and without the presence of a metabolic activation system, were comparable to those observed in the vehicle control group, as per the criteria employed for evaluation of mutagenic potential, and this observation was confirmed by repetition of the experiments. Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX. They also compared well with the range reported in the literature. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation.

Conclusions

Under the conditions of this study, the test item is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.