L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

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:

21 June - 05 July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted under GLP in accordance with the international guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

EC Number:

843-143-1

Cas Number:

709647-81-4

Molecular formula:

C9H21ClN4O4

IUPAC Name:

L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

Test material form:

liquid

Remarks:

Clear, colourless

Details on test material:

Sponsor's substance name ID: N-glyceryl arginine HCl
Storage conditions ~4 ºC, in the dark

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg per plate) where no growth inhibition was observed in all tested strains with and without metabolic activation a. Precipitation was also not observed in all strains with and without metabolic activation. Therefore the doses for the main test were as follows;
0, 50, 150, 500, 1500 & 5000 μg per plate for all tested strains with or without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile water
- Justification for choice of solvent/vehicle: guideline recommended

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA), 1 μg per plate for TA100, 2 μg per plate for TA1535/37 & 10 μg per plate for WP2uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate
- Number of independent experiments: Three ; preliminary test and two main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): n/a
- Test substance added in medium; Direct plate incorporation was used for the assay. Measured aliquots (0.1 ml) of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicates for each bacterial strain and for each concentration of test material both with and without S9-mix. All plates were incubated at at 37 °C for 48 hours and the frequency of revertant colonies assessed using Domino colony counter.
The second experiment was performed using dose range of 50 – 5000 ug/plate. The assay was perfomed by mixing 0.1 ml of bacterial culture, 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar plates (30 ml/plate). In addition, 0.1 ml of the maximum concentration of test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for revertant colonies using Domino colony counter.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: N/A
- Exposure duration/duration of treatment: 48 hours at 37 °C
- Harvest time after the end of treatment (sampling/recovery times): not stated

Rationale for test conditions:

The reverse mutation was considered valid if the following criteria are met;
All tester strains cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
there should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plate due to contamination.
All tester strain cultures should be in the approximate range of 1 – 9.9 x109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the result will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significant will not be the only determining factor for positive response.
A test material will be considered non-mutagenic (negative in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgment about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Not stated

Results and discussion

Test results

Test results 1

Key result

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:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

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:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Key result

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:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Key result

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:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

MTEST-SPECIFIC CONFOUNDING FACTORS: 
- Data on pH: Not specified
- Data on osmolality: Not specified
- Possibility of evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation and time of the determination: No precipitation was observed at all doses with and without metabolic activation.
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no



RANGE-FINDING/SCREENING STUDIES (if applicable): Was conducted, no growth inhibition was observed in all TA strains with and without metabolic activation and Precipitation was not observed in all groups with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Both with historical control range


For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: No increase in revertant were observed.
- Statistical analysis; not stated

- Any other criteria: not stated


Remarks on result:

other: Negative for mutagenicity

Applicant's summary and conclusion

Conclusions:

Based on the conditions of the study, the test item did not induce mutagenicity in the bacterial reverse mutation assay with or without metabolic activation.

Executive summary:

OECD 471 ( 2005 ): The test item, was tested to evaluate its mutagenic potential using the preincubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence and absence of an exogenous metabolic activation system. Distilled water was used as the vehicle.

 

The doses for the main test were as follows; 0, 50, 150, 500, 1500  & 5000 μg per plate for all tested strains with or without metabolic activation were selected based on the result of the preliminary study.

The test item in distilled water at, at concentrations of 50 mg/mL, was stable at room temperature under the condition of the study. No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 mixes.

 

Both positive and negative control gave acceptable results, confirming the validity of the test system. No visible reduction in the growth of bacterial background lawn or precipitate were observed with all tested strains under all conditions. No dose dependent increases were in all tester strain with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.

 

Under the conditions of this study, test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.