.beta.-D-Ribofuranose, 5-deoxy-, 1,2,3-triacetate

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:

13 September 2012 to 14 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and in accordance with current test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetylfuranoside

IUPAC Name:

Acetylfuranoside

Constituent 2

Reference substance name:

ß-D-Ribofuranose, 5-deoxy-, triacetate (6CI,9CI)

EC Number:

612-957-7

Cas Number:

62211-93-2

Molecular formula:

C11 H16 O7

IUPAC Name:

ß-D-Ribofuranose, 5-deoxy-, triacetate (6CI,9CI)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: Acetylfuranoside (also known as ß-D-Ribofuranose, 5-deoxy-, 1,2,3-triacetate)
CAS number: 62211-93-2
Batch number: 201203272009
Quantity received: 1.2 kg
Purity: 98.7%
Retest date: 31 Mar 2013
Date of receipt: 14 September 2012
Storage details: When not in use the test item was stored in a sealed container, at room temperature, in the dark.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

For all assays, bacteria were cultured at 37±1°C for 7 or 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell count data from testing of each strain batch

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Mutation experiment 1 (with and without S-9)
Concentration of treatment solution 0.05000 mg/mL, final concentration 5.000 µg/plate
Concentration of treatment solution: 0.1581 mg/mL, final concentration: 15.81 µg/plate
Concentration of treatment solution: 0.5000 mg/mL, fnal concentration: 50.00 µg/plate
Concentration of treatment solution: 1.581 mg/mL, final concentration: 158.1 µg/plate
Concentration of treatment solution: 5.000 mg/mL, final concentration: 500.0 µg/plate
Concentration of treatment solution: 15.841 mg/mL, final concentration: 1581 µg/plate
Concentration of treatment solution: 50.00 mg/mL, final concentration: 5000 µg/plate

Mutation experiment 2 (with and without S-9)
Concentration of treatment solution: 1.563 mg/mL, final concentration: 156.3 µg/plate
Concentration of treatment solution: 3.125 mg/mL, final concentration: 312.5 µg/plate
Concentration of treatment solution: 6.250 mg/mL, final concentration: 625.0 µg/plate
Concentration of treatment solution: 12.50 mg/mL, final concentration: 1250 µg/plate
Concentration of treatment solution: 25.00 mg/mL, final concentration: 2500 µg/plate
Concentration of treatment solution: 50.00 mg/mL, final concentration: 5000 µg/plate

Positive controls
2-nitrofluorene (2NF), stock concentration: 50 µg/mL, Final concentration: 5 µg/plate, TA98 without S-9
Sodium azide (NaN3), stock concentration: 20 µg/mL, Final concentration: 2 µg/plate, TA100 and TA1535 without S-9
9-aminoacridine (AAC), stock concentration: 500 µg/mL, Final concentration: 50 µg/plate, TA1537, without S-9
Mitomycin C (MMC), stock concentration: 2 µg/mL, Final concentration: 0.2 µg/plate, TA102, without S-9
Benzo[a]pyrene (B[a]P), stock concentration: 100 µg/mL, Final concentration: 10 µg/plate, TA98, with S-9
2-aminoanthracene (AAN), stock concentration: 50 µg/mL, Final concentration: 5 µg/plate, TA100, TA1535 and TA1537, with S-9
2-aminoanthracene (AAN), stock concentration: 200 µg/mL, Final concentration: 20 µg/palte, TA102, with S-9

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Remarks:

For concentrations see test concentrations section

Details on test system and experimental conditions:

Acetylfuranoside was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, before the addition of 2.5 mL molten agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Acetylfuranoside at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 - 5000 µg/plate, in order to examine more closely those concentrations of Acetylfuranoside approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p < 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Dunnett's test

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Acetylfuranoside at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, no evidence of toxicity was observed, as would normally be manifest by a diminution of the background bacterial lawn, and/or a marked reduction in revertant numbers.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 - 5000 µg/plate, in order to examine more closely those concentrations of Acetylfuranoside approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.
The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Mutation results
Following treatments of all the test strains in the absence and presence of S-9, only Experiment 1 treatments of strain TA102 in the presence of S-9 at 1581 µg/plate resulted in an increase in revertant numbers that was statistically significant when the data were analysed at the 1% level using Dunnett’s test. The increase was small in magnitude and occurred at a single intermediate concentration with no indication of a concentration relationship. Accordingly this increase was considered to have been due to normal biological variability and not evidence of mutagenic activity.
No other increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no clear evidence of any Acetylfuranoside mutagenic activity in this assay system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that Acetylfuranoside did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Acetylfuranoside was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Acetylfuranoside treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Acetylfuranoside at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments no evidence of toxicity was observed.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 mg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 - 5000 µg/plate, in order to examine more closely those concentrations of Acetylfuranoside approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Although a, statistically significant increase in revertant numbers was observed following Experiment 1 Acetylfuranoside treatments of strain TA102 at 1581 µg/plate in the presence of S-9 (Dunnett's Test, 1% level), this was not concentration-related or reproducible and was of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system.

It was concluded that Acetylfuranoside did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).