N-acetyl-β-D-glucosamine

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:

July 8th 2019 - November 9th 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Bacterial Reverse Mutation Test - Salmonella typhimurium

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-acetyl-β-D-glucosamine

EC Number:

231-368-2

EC Name:

N-acetyl-β-D-glucosamine

Cas Number:

7512-17-6

Molecular formula:

C8H15NO6

IUPAC Name:

N-acetyl-β-D-glucosamine

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form the “S9 mix”. This mix is added to the top agar in this activated assay. The S9 fraction procured from M/S G.P. Meshram, Nagpur, and (Lot N ° MWR/ARI/S9F/ 01/18, APPENDIX 7) was used in the study.

Co-factor Mix

D- Glucose –6- phosphate: 0.80 g
beta-Nicotinamide adenine dinucleotide Phosphate (beta-NADP): 1.75 g
Magnesium chloride: 0.90 g
Potassium chloride: 1.35 g
Sodium phosphate, dibasic: 6.40 g
Sodium phosphate, monobasic: 1.40 g
Distilled water: 450 mL

Constituent 5% v/v S9 mix (10 mL) 10% v/v S9 mix (10 mL)
Co-factor mix 9.5 mL 9.0 mL
S9 fraction 0.5 mL 1.0 mL

Test concentrations with justification for top dose:

An initial toxicity test was performed and showed no increase in the number of revertant colonies observed at 5000 µg N-acetyl-D-glucosamine /plate with and without S9 activation at 5% v/v S9 mix. Based on these results six concentrations 156.25, 312.5, 625, 1250, 2500 and 5000 µg N-acetyl-D-glucosamine /plate, in the absence and presence of the metabolic activation system (10% v/v S9 mix) for TA1537, TA1535, TA98, TA100, and TA102, was selected for the confirmatory mutation test.

Vehicle / solvent:

Distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Details on test system and experimental conditions:

Tester strains TA1537, TA1535, TA98, TA100, and TA102 were exposed to test concentrations of 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate of N-acetyl-D-glucosamine both in the absence and presence of metabolic activation (10 % v/v S9 mix). Plates were maintained in triplicates for each test concentration of test item, negative and positive controls. The numbers of revertant colonies were recorded after 48 h incubation period at 37 ± 1 ºC.

Rationale for test conditions:

Treatment with 2-aminoanthracene in the absence of the metabolic activation system was also performed for tester strain TA100 in both the initial toxicity-mutation test and the confirmatory mutation test to verify the efficiency of the S9 fraction used in the study. Since 2-aminoanthracene should not be used as the sole indicator of S9 mix, by testing this control in the absence of S9 it demonstrates that the chemical requires metabolic activation by S9 in order to be mutagenic in this test system.

Evaluation criteria:

A result is considered positive if 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. Biological relevance of the result was considered first.
Strains TA1535, TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strains TA98, TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

Statistical analysis was used as an aid in the evaluation of dose response.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second experiment, using the same method as specified above, with an alteration in concentration spacing and S9 concentration.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of revertant colonies.

Results and discussion

Test results

Test results 1

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate)

Test results 2

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Sodium Azide (0.5 µg/plate)

Test results 3

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-Nitrofluorene (7.5 µg/plate)

Test results 4

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Sodium Azide (5 µg/plate)

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Mitomycin-C (0.5 µg/plate)

Additional information on results:

All values determined in the negative control tests were within historical controls for their respective strains. An increase in revertants was not observed in tester strain TA100 (in both the initial and confirmatory mutation test) when treated with 2-aminoanthracene in the absence of the metabolic activation but a clear increase in revertant numbers was observed in the presence of the metabolic activation. This demonstrated the efficiency of the S9, fraction used in this assay. All positive controls exhibited clear increases in the number of revertant colonies when compared with the negative controls thus are considered valid. Cytotoxicity was not observed as the background bacterial lawn and numbers of revertant colonies showed no treatment related effects for the test item up to 5000 µg/plate. No significant increase in revertant colonies was observed for any test strain with or without metabolic activation when treated with the test item up to 5000 µg/plate when compared to the concurrent negative controls and all results were within the historical control ranges.

Applicant's summary and conclusion

Conclusions:

A normal bacterial background lawn, without reduction in number of revertant colonies was observed up to 5000 µg/plate in TA1537, TA1535, TA98, TA100 and TA102, in the absence and presence of the metabolic activation system (10% v/v S9 mix). From results of this study, it is concluded that N-acetyl-D-glucosamine is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under specified experimental conditions.