Sodium glucoheptonate

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:

The experimental phase of this study was performed between 22 February 2012 and 05 November 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

Sodium glucoheptonate

EC Number:

250-480-2

EC Name:

Sodium glucoheptonate

Cas Number:

31138-65-5

Molecular formula:

C7H14O8.Na

IUPAC Name:

disodium (2R,3R,4S,5R,6R)-2,3,4,5,6,7-hexahydroxyheptanoate (2S,3R,4S,5R,6R)-2,3,4,5,6,7-hexahydroxyheptanoate

Test material form:

other: liquid

Details on test material:

Sponsor's identification: Sodium Glucoheptonate
Description: dark brown liquid
Lot number: 921000100
Purity: 49.5%
Date received: 09 February 2012
Expiry date: 13 January 2013
Storage conditions: room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor frame shift mutations
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor base-pair substitutions


All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)).

The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, model SXR 34.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

E. coli
Strain Genotype Type of mutations indicated
WP2uvra trp-;uvrA base-pair substitutions
In addition to a mutation in the tryptophan operon, the
E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel (1976)).

The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, model SXR 34.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Mutation Test - Experiment 1
Five concentrations of the test item (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Mutation Test - Experiment 2:The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was the same as Experiment 1 (50 to 5000 µg/plate).

Vehicle / solvent:

The test item was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs from the British Industrial Biological Research Association, on nutrient agar plates. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Mutation test - Experiment 1:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the vehicle, test item formulation 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 triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using an automated colony counter.

Mutation test - Experiment 2:
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was the same as Experiment 1 (50 to 5000 µg/plate).
As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using an automated colony counter.

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: yes, but period not stated
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency

OTHER EXAMINATIONS:
Preliminary toxicity test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test item formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test item formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item 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 making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

Tables 1 to 5 can be found in the attached background information section.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item, Sodium Glucoheptonate, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Sodium Glucoheptonate, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver 89 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 to 5000 ~g/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

Results:

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion:

The test item, Sodium Glucoheptonate, was considered to be non-mutagenic under the conditions of this test.