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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic potential

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is detailed in the report attached to the IUCLID section 13.

Principles of method if other than guideline:

Reverse mutation assays using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki (1975).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, TA98

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsome fraction (S9)

Test concentrations with justification for top dose:

Six concentrations; maximum dose tested of 5000 µg/plate.

Vehicle / solvent:

- Vehicle used: phospahte buffer.

Untreated negative controls:

yes

Remarks:

The experiment was carried out on 190 synthetic food additives and 52 food additives derived from natural sources. Both positive and negatve results were recorded.

Details on test system and experimental conditions:

DURATION
Preincubation period: cell cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37 °C before plating.

NUMBER OF REPLICATIONS
Duplicate plates were used for each of six different concentrations of the sample.

EXAMINATION
The number of revertant (his +) colonies was scored after incubation at 37 °C for 2 days.

S9 MIX
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip).
The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 mM-NADPH, 4 mM-NADH, 33 mM-KC1, 8 mM-MgCI 2, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml.

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

Key result

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

No significant increases in the number or revertant colonies were detected in any S. typhimurium strains at the maximum dose.

Conclusions:

The substance did not induce gene mutations in Salmonella typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98, with and without metabolic activation.

Executive summary:

Method

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum. The following strains were used: TA92, TA1535, TA100, TA1537, TA94 and TA98 . The test was performed with and without the addition of supernatant (S9 fraction). The compound was dissolved in phosphate buffer and tested at six concentrations, up to 5000.0 µg/plate in the presence and absence of a metabolic activation system.

The experiment was carried out on 190 synthetic food additives and 52 food additives derived from natural sources. Both positive and negatve results were recorded.

Results

The substance did not induce gene mutations in Salmonella typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98, with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is detailed in the report attached to the IUCLID section 13.

Principles of method if other than guideline:

Chromosomal aberration tests were carried out using a Chinese hamster fibroblast cell line, CHL, as described elsewhere (Ishidate & Odashima, 1977).

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10 % calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr.

Metabolic activation:

without

Test concentrations with justification for top dose:

Three dosed; the maximum dose tested 1 mg/ml

Vehicle / solvent:

- Vehicle: physiological saline solution.

Untreated negative controls:

yes

Remarks:

The experiment was carried out on 190 synthetic food additives and 52 food additives derived from natural sources. Both positive and negatve results were recorded.

Details on test system and experimental conditions:

EXPOSURE
The cells were exposed to each sample at three different doses for 24 and 48 hr.

CHROMOSOME PREPARATION
Colcemid (final concn 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5 %, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens).

EXAMINATION
The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.

TEST CONCENTRATION
The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50 % cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).

Evaluation criteria:

The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels.

For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20 % of the metaphases observed. In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/ml). TR values are relatively high for chemicals that show carcinogenic potential in animals (Ishidate, Sofuni & Yoshikawa, 1981).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

all strains/cell types tested

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Remarks:

the incidence of aberrations was usually less than 3.0 %

Additional information on results:

Incidence of polyploid cells: 0 % at 48 hrs
Structural aberration: 1.0 % at 48 hrs

Conclusions:

The substance did not induce chromosome aberration in Chinese hamster fibroblast cell line.

Executive summary:

Method

The substance was tested for mutagenic effects in vitro in using a Chinese hamster fibroblast cell line. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo. In the present studies, no metabolic activation systems were applied.

Untreated cells and solvent-treated cells served as negative controls. The experiment was carried out on 190 synthetic food additives and 52 food additives derived from natural sources. Both positive and negatve results were recorded.

Results

Based on the results of these experiments, it is concluded that tested substance did not induce chromosome aberration in Chinese hamster fibroblast cell line.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is detailed in the report attached to the IUCLID section 13.

Principles of method if other than guideline:

Sister chromatid exchange Human peripheral blood Lymphocytes.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 10, 50 and 100 µg/ml

Vehicle / solvent:

Saline solution

Untreated negative controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION
The whole-blood microculture technique was used. 0.4 ml of venous whole blood was inoculated to 5 ml of the culture medium.

CULTURE MEDIUM
- Composition: containing
41.6 mg of RPMI 1640 (JR Scientific Inc., Woodland, CA)
1 ml of fetal calf serum (purchased from Tianjin Medical College, China)
1.5 mg of PHA (produced by Guangdong Biological Product Institute, China)
10000 IU each of penicillin and streptomycin
0.1 ml of 5 % NaHCO3.
- pH: 7

INCUBATION
The mixture was incubated for 24 h. Bromodeoxyuridine (BrdU), at 10 µg/ml, was added at 24 h of culture.
At 48 h of culture, substance (dissolved in saline solution) or saline solution (for control) was added and the cultures were incubated for another 24 h.

HARVESTING
Peripheral Blood Lymphocytes (PBL) were harvested at the end of the 24 h exposure period (total culture time = 72 h) with 0.05 µg/ml colchicine being added 2 h before harvesting.
All cultures were incubated at 37 °C in the dark.
PBL, harvested after gentle centrifugation (1000 rpm, 10 min) and discarding the supernatant, were resuspended in 0.075 M KCI solution for hypotonic treatment for 20 min, centrifuged again, and finally fixed in methanolacetic acid solution (3:1) three times.

SLIDES PREPARATION
A few drops of the cell suspension were dropped onto a clean slide taken from icy water and frame-dried. The slides were incubated in 2 × SSC solution in petri dishes at 70 °C and irradiated simultaneously under a UV lamp (30 W) at a distance of 10 cm for 20 min. The preparations were stained in 3 % Giemsa solution for 10 min after being rinsed with tap water (Xing, 1990; Zhang, 1991).

SCE ANALYSIS
SCE analysis was carried out on cells having replicated for two cycles in the presence of BrdU. Ten metaphases were scored for each treatment.

Statistics:

Statistical methods for analysis of variance (AOV) based on the square roots of SCE were used (Crossen, 1977; Morgan, 1977, 1981; Vercauteren, 1984)

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Additional information on results:

After multiple comparisons, the effects of test article on SCE in PBL are not significant.

Further substances were involved in the test and divided into two groups with each group consisting of five substances. A randomized complete-block design was used to avoid the influence of individual differences on SCE. One person's blood was used for each test and regarded as one block.

Remarks on result:

other: no mutagenic potential

 

 

Conclusions:

No mutagenic potential

Executive summary:

Effects of Target substance on SCE in PBL

The experimental data show that the treatment with test substance did not induce a significant increase in SCE. The test item is an essential substance for PBL growth. Thus, it was unexpected that the target substance can significantly induce SCE in PBL. We infer that the addition of exogenous substances might result in an imbalance among tested substances in the medium and cause metabolic disturbances in cells. This might further influence the activities of various enzymes and result in the induction of SCE.

However, this is only a hypothesis and the actual molecular mechanisms involved remain unsolved.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: official EFSA document, only short abstract available.

Justification for type of information:

Justification for Read Across is detailed in the report attached to the IUCLID section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

not specified

Type of assay:

other: In vitro gene mutation assay in mammalian cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The maximum tested concentration was 6500 µg/plate.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

No evidence of mutagenicity observed.

Executive summary:

Method

Gene mutation tests (in accordance with OECD Guideline 476) at the TK locus of the L5178Y cell line were performed, in both the presence and absence of metabolic activation, with concentrated liquid L-lysine HCl at doses up to 6500 µg/ml.

Results

No evidence of mutagenicity was found.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The assessment of the endpoint is performed with the integrated evaluation of results for in vitro bacteria gene mutation, mammalian cell gene mutation and chromosomal aberration assays andin vivomicronucleus assay. The substance is expected to be biochemically stable and not bioavailable; it is expected to be mainly excreted as such, without interaction with the life-system. Because of the possibility of a partial metabolism/transformation of the substance cannot be excluded, the available information on possible precursors, which could be also the metabolites/transformation products of the target substance, has been precautionary taken into account. The read across approach can be considered as reliable and appropriate for the endpoint under investigation; details on the read across approach are included in the reportattached to the IUCLID section 13.

The test were conducted on the substance in itself (in vitro bacteria gene mutation) and on the metabolites/transformation products of the target substance (mammalian cell gene mutation and chromosomal aberration assays and in vivo micronucleus assay).

 

One test (in vitro chromosomal aberration) conducted on the disodium succinate (RA Substance 1b) gave equivocal results, nevertheless another test of chromosomal aberration conducted on Succinic acid (RA Substance 1a) is available and gave a negative result, thus the equivocal test was disregarded and considered, for the chemical safety assessment, the succinic acid test only. All the other available tests showed negative results.

No relevant differences occurred in the mutagenicity studies with and without the metabolising system.

 

During In vivo study, which is the study most representative of the behaviour of the chemicals in a living being, there was no indication of an increase in micronuclei under the test conditions.

 

Considering all the available information the substance can be considered as non genotoxic.

The evaluation of the single mutagenicity endpoint as required by the REACH Regulation (EC n. 1907/2006) was conducted as follow:

IN VITRO BACTERIA REVERSE MUTATION ASSAY -AMES TEST

Target substance

[Raval (2009). Set Retard Plus - Salmonella typhimurium, Reverse Mutation Assay (Ames Test). Testing laboratory: Intox Pvt Ltd. Report N. 9656. Owner company: Prosol S.p.A.]

Salmonella typhimurium, Reverse Mutation Assay of Set Retard Plus was carried out following the OECD Guidelines for Testing of Chemicals (No. 471). TA1535, TA97a, TA98, TA100 and TA102 strains were assayed, in the presence and absence of metabolic activation system (S9), at the following concentrations: 5000, 1500, 500, 150 and 50 µg/plate. Results indicated that the frequencies of histidine revertant colonies at all concentrations of test item in all strains, with and without the presence of a metabolic activation system, were comparable to those observed in the vehicle control group. Thus, under the conditions described for the study, it is concluded that, Set Retard Plus is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.

Negative

 

RA Substance 1a (succinic acid)

[Ishidate et al. (1984); Primary Mutagenicity Screening Of Food Additives Currently Used In Japan; Fd Chem. Toxic. Vol. 22, No. 8, pp. 623-636]

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum, using the strains: TA92, TA1535, TA100, TA1537, TA94 and TA98 . Six concentrations in the range of 0.1 to 5000.0 mg/plate in the presence and absence of a metabolic activation system were tested. Based on the results of these experiments, it is concluded that tested substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Negative

RA Substance 1d (disodium succinate)

[Ishidate et al. (1984); Primary Mutagenicity Screening Of Food Additives Currently Used In Japan; Fd Chem. Toxic. Vol. 22, No. 8, pp. 623-636]

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum, using the strains: TA92, TA1535, TA100, TA1537, TA94 and TA98. Six concentrations in the range of 0.1 to 5000.0 mg/plate in the presence and absence of a metabolic activation system were tested. Based on the results of these experiments, it is concluded that tested substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Negative

RA Substance 2b (lysine hydrochloride)

[EFSA (2013); Scientific Opinion on the safety and efficacy of concentrated liquid L-lysine (base), concentrated liquid L-lysine monohydrochloride and L-lysine monohydrochloride produced by Escherichia coli (FERM BP-10941) for all animal species, based on three dossiers submitted by Ajinomoto Eurolysine SAS; EFSA Journal;11(10):3365]

Data from secondary source are available on lysine hydrochloride. Unfortunately there are no many details about testing procedures and results; the information is in any case here mentioned for completeness sake.

Bacterial reverse mutation assays were carried out, in compliance with OECD Guideline 471, on four S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and E. coli WP2uvrA with and without metabolic activation. The maximum tested concentration was 5000 µg/plate. No evidence of mutagenic activity was observed.

Negative

IN VITRO MAMMALIAN CELL GENE MUTATION

RA Substance 2b (Lysine hydrochloride) 

[EFSA (2013); Scientific Opinion on the safety and efficacy of concentrated liquid L-lysine (base), concentrated liquid L-lysine monohydrochloride and L-lysine monohydrochloride produced by Escherichia coli (FERM BP-10941) for all animal species, based on three dossiers submitted by Ajinomoto Eurolysine SAS; EFSA Journal;11(10):3365]

Data from secondary source are available on lysine hydrochloride. Gene mutation tests were performed on the TK locus of the L5178Y cell line, in accordance with OECD Guideline 476. The experiments were carried out both the presence and absence of metabolic activation, with concentrated liquid test item at doses up to 6500 µg/ml. No evidence of mutagenicity was found.

Althought details about the substance purity and composition are not given, the information in the publication is regarded as reliable; indeed, because of the manufacture process, the substance is expected to possess an appreciable purity degree.

Negative

IN VITRO CHROMOSOMAL ABERRATION

RA Substance 1a (succinic acid)

[Ishidate et al. (1984); Primary Mutagenicity Screening Of Food Additives Currently Used In Japan; Fd Chem. Toxic. Vol. 22, No. 8, pp. 623-636]

The substance was tested for mutagenic effects in vitro in using a Chinese hamster fibroblast cell line with no metabolic activation systems applied. Untreated cells and solvent-treated cells served as negative controls. The experiment was carried out on 190 synthetic food additives and 52 food additives derived from natural sources. Both positive and negatve results were recorded.

Based on the results of these experiments, it is concluded that tested substance did not induce chromosome aberration in Chinese hamster fibroblast cell line.

Negative

RA Substance 1d (disodium succinate)

[Ishidate et al. (1984); Primary Mutagenicity Screening Of Food Additives Currently Used In Japan; Fd Chem. Toxic. Vol. 22, No. 8, pp. 623-636]

The substance was tested for mutagenic effects in vitro in using a Chinese hamster fibroblast cell line. The cell line was originally established from the lung of a newborn female. No metabolic activation systems were applied. Untreated cells and solvent-treated cells served as negative controls. The experiment was carried out on 190 synthetic food additives and 52 food additives derived from natural sources. Both positive and negative results were recorded.

The experiment gave equivocal results, thus the test has been disregarded; this information has been included into the dossier for completeness sake.

Equivocal 

IN VITRO SISTER CHROMATID EXCHANGE ASSAY

RA Substance 2a (lysine)

[Zhang and Yang (1992). Effects of amino acids on sister-chromatid exchanges. Mutat. Res. 280(4), 279-283]

The he effects of Lysine on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) was studied. The substance did not induced a significant increase in SCE in PBL. SCE analysis was carried out on cells having replicated for two cycles in the presence of BrdU, at the concentrations of 10, 50 and 100 µg/ml. Ten metaphases were scored for each treatment. After multiple comparisons, the effects of test article on SCE in PBL resulted to be not significant.

Negative

IN VIVO CHROMOSOMAL ABERRATION

RA Substance 2b (lysine hydrochloride)

[EFSA (2013); Scientific Opinion on the safety and efficacy of concentrated liquid L-lysine (base), concentrated liquid L-lysine monohydrochloride and L-lysine monohydrochloride produced by Escherichia coli (FERM BP-10941) for all animal species, based on three dossiers submitted by Ajinomoto Eurolysine SAS; EFSA Journal;11(10):3365]

Data from secondary source are available on lysine hydrochloride. Unfortunately there are no many details about testing procedures and results. An in vivo micronucleus test study was conducted in accordance with OECD 474 using Wistar rats at doses up to 6 %, in connection with a 13-weeks subchronic oral toxicity study by means of bone marrow cells harvested from the femur at necroscopy. There was no indication of an increase in micronuclei under the test conditions.

Negative

CONCLUSION

Based on all the available data, Set Retard Plus is not expected to be genotoxic. Although, in some cases, the available information are not well detailed, the totality of the available data agree to not identify reasons of concern.

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

 

Due to the results available on the target substance and on his metabolites, the substance can be stated as non genotoxic.

 

In conclusion, the substance is not classified for genetic toxicity according to the CLP Regulation (EC n. 1272/2008).