α-(1→2) branching sucrase

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:

September 15, 2017 - March 20, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 2

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Active enzyme protein of Dextransucrase (CAS no. 9032-14-8, Enzyme Class no. 2.4.1.5)

Molecular formula:

Not applicable, please see 'Remarks'

IUPAC Name:

Active enzyme protein of Dextransucrase (CAS no. 9032-14-8, Enzyme Class no. 2.4.1.5)

Test material form:

liquid

Details on test material:

- Substance type: UVCB
- Name of test material (as cited in study report): H-32217
- Lot/batch No.: 20178041
- Physical state: Clear brown liquid

Method

Target gene:

Histidine locus in the genome of Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, and at the tryptophan locus of the Escherichia coli strain WP2 uvrA

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: Eight concentrations of the test item (33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 ug total protein/plate) in the absence and presence of S9 metabolic activation
Experiment 2: Five concentrations of the test item (333, 667, 1000, 3333, and 5000 μg total protein/plate in the presence of S9 activation and 1, 10, 100, 1000, and 5000 μg total protein/plate for all tester strains in the absence of S9 activation.
The highest dose of 5000 μg is equivalent to 3.294 mg active enzyme protein/ kg bw or 8.197 enzyme concentrate dry matter/kg bw.

Vehicle / solvent:

sterile water

Controls

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other:

Details on test system and experimental conditions:

Preparation and Storage of Tester Strain: Frozen permanent stocks of all tester strains were prepared by growing fresh cultures and adding 0.09 mL DMSO per milliliter of culture. Aliquots were frozen in dry ice and stored at ≤-70°C. Master plates were prepared by streaking each tester strain from a frozen permanent stock onto either nutrient agar plates or minimal glucose agar plates. The minimal glucose agar plates were supplemented with either histidine and biotin or tryptophan, and for strains containing the pKM101 plasmid, ampicillin. Tester strain master plates were stored at 5 ± 3°C. Cultures for use in the study were inoculated from the appropriate master plates. The cultures were placed in a shaker/incubator for overnight at 100 to 200 rpm and 37 ± 2°C. To ensure that appropriate numbers of bacteria are plated, the length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested when the tester strain culture titers were approximately 109 cells per millimeter.
Experimental Design and Methodology:
-Solubility Determination and Selection of Vehicle: A solubility determination was not conducted. Based on the nature of the test article and compatibility with the target cells, sterile water was chosen as the test article solvent.
-Exogenous Metabolic Activation and Sham Mix: Liver homogenate (S9), prepared from male Sprague-Dawley rats that have been induced with Aroclor 1254 were purchased and stored frozen at approximately -80ºC until used. The S9 was thawed and the 10% S9 mix prepared immediately prior to its use. The S9 mix was held on ice at all times.
-Sterility Controls : The most concentrated test substance dilution was checked for sterility by plating 100 μL on selective agar plates. The S9 mix, sham mix, and re-suspension broth were checked for sterility by plating 0.5 mL on selective agar plates.
-Plate Identification, Frequency, and Route of Administration: This test was conducted using the treat and plate modification of the pre-incubation method. This method has an additional step to remove the article from the bacteria prior to plating on the selective media. This method has been selected due to the potential of the test article to interfere with the selective conditions of the assay, potentially leading to false positive results. Each plate was labeled as required. The plate number signified a positive control, a negative control or a sample plate, and tester strain, the absence or presence of S9 metabolic activation, test article concentration, and replicate.
Each treatment tube was labeled with the appropriate plate number(s) that correspond to the tube contents. The total protein content of the neat test article is less than 50 mg/ml. Therefore, the volume of the treatment mixture in each pre-incubation tube was increased accordingly, in order to ensure testing up to the OECD Test Guideline 471 recommended highest concentration of 5000 μg/plate. Each tube was capped and incubated at approximately 37°C and 150 rpm, for a 60-minute treatment period. At the end of this treatment period the treatment mixture was transferred to a centrifuge.
Treatment tubes were centrifuged for 10 minutes at 1500 rcf. The supernatant was aspirated down to ~50 μL. The pelleted bacteria were re-suspended with enough nutrient broth to match the original volume of bacterial culture. Plates were prepared by transferring 100 μL of re-suspended bacteria to a pre-heated (45-48°C) glass culture tube containing 2.5 mL of selective top agar. The bacteria were mixed with the agar by vortexing and then overlaid onto the surface of minimal glucose agar plates.
The positive control for WP2 uvrA in the presence of S9 activation was tested using the plate incorporation method. One hundred 100 μL of the positive control was added to pre-heated (45-48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 μL of tester strain and 0.5 mL of S9 mix. The mixture was vortexed and overlaid onto the surface of a minimum glucose agar plate.
After the overlay solidified, the plates were inverted and incubated for approximately 48 to 66 hours at 37 ± 2°C. Plates that were not counted immediately following the incubation period were stored at 5 ± 3°C. All toxicity-mutation test dose preparations of negative (vehicle) controls, test article, and positive controls were plated in duplicate. All mutagenicity test dose preparations of negative (vehicle) controls, test article, and positive controls were plated in triplicate.
Scoring: The appearance of the bacterial background lawn was assessed microscopically for test article toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered a sign of toxicity. Plates with observation of background lawn toxicity or precipitation were assigned a toxicity or precipitation code. Background lawn toxicity and precipitation were documented by exception only; no codes were assigned to plates with normal background lawn and that were free of precipitation.
Revertant colonies were counted with the Sorcerer automated colony counter using Ames Study Manager software (Perceptive Instruments Ltd., Suffolk, United Kingdom). Plates that could not be accurately counted automatically were counted manually.

Evaluation criteria:

Criteria for a positive response:
1. Strains TA1535 and TA1537
Data sets will be judged positive if the increase in mean revertant colonies at the highest numerical dose response is ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test article unless observed at the top concentration only.
2. Strains TA98, TA100 and WP2 uvrA
Data sets will be judged positive if the increase in mean revertant colonies at the highest numerical dose response is ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test article unless observed at the top concentration only.
A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response will be evaluated as negative if it is neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 100 μg/plate however all background lawn reductions observed were microscopic. No reduction in the number of revertant colonies was observed.

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 100 μg/plate however all background lawn reductions observed were microscopic. No reduction in the number of revertant colonies was observed.

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 100 μg/plate however background lawn reduction in the top two dose levels was visible by eye but the third dose level was a microscopic reduction. No reduction in the number of revertant colonies was observed

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 100 μg/plate however background lawn reduction in the top two dose levels was visible by eye but the third dose level was a microscopic reduction. No reduction in the number of revertant colonies was observed

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

staring at 1000 μg/plate

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 9

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 10

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

A minimum of 3 non-toxic scorable test article concentrations are required to validate the study. A test article concentration is considered toxic if it causes:
• A >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibits a concentration-dependent drop in the revertant count, or
• A reduction in the background lawn.
Less than three of the concentration levels scored were non-toxic for tester strains TA98, TA100, TA1535 (toxicity-mutation test only), and TA1537 in the absence of S9 activation. However, the toxicity was observed as background lawn reduction only. No reduction in the number of revertant colonies was observed. It was decided that additional testing at lower concentrations was not necessary to make a mutagenicity assessment in this test.

Applicant's summary and conclusion

Conclusions:

The test article (H-32217) is not mutagenic in the Ames assay in both the presence and absence of metabolic activation.

Executive summary:

The objective of this assay was to assess the potential of the test article (H-32217) to induce mutagenicity in the Bacterial Reverse Mutation Test using the treat and plate modification of the pre-incubation method in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2 uvrA in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The study was conducted according to the OECD Guideline 471 in compliance with GLP. 

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the concentration range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test article.

All test article concentrations were prepared based on the total protein concentration. The neat test article was the highest concentration used on this study. All lower concentrations were formulated in sterile water. All reported dose concentrations represent the amount of total protein per unit.

In the toxicity-mutation test, the maximum concentration evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvrA in the absence and presence of S9 metabolic activation. This concentration was achieved using the neat test article and a 179 μL treatment aliquot. All tester strains and test conditions were evaluated at 8 concentration levels along with the negative (vehicle) and positive controls. The concentration used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. The highest dose is equivalent to 3.294 mg active enzyme protein/ kg bw or 8.197 enzyme concentrate dry matter/kg bw.

No positive mutagenic responses were observed at any concentration level in any tester strain in the absence or presence of S9 metabolic activation. No test article precipitation was observed. Toxicity was observed a background lawn reduction in all tester strains in the absence of S9 activation starting at 100, 66.7, 333, 33.3, and 333 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvrA, respectively. A reduction in background lawn was also observed in the presence of S9 activation with tester strain WP2 uvrA starting at 3333 μg/plate. Although there were less than three non-toxic dose levels scored for tester strains TA98, TA100, and TA1537 in the absence of S9 activation, the toxicity was observed as background lawn reductions only, with no concentration related affect, and no reduction in the number of revertant colonies. It was determined that testing at lower concentrations was not necessary for a preliminary mutagenicity assessment and selection of concentration ranges for the main mutagenicity test.

Based on the toxicity-mutation test, the maximum concentration evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2 uvrA in the absence and presence of S9 metabolic activation. This concentration was achieved using the neat test article and a 179 μL treatment aliquot. All tester strains and test conditions were evaluated at 5 concentration levels along with the negative (vehicle) and positive controls. The concentrations used in this test were 333, 667, 1000, 3333, and 5000 μg/plate for all tester strains in the presence of S9 activation and 1, 10, 100, 1000, and 5000 μg/plate for all tester strains in the absence of S9 activation. No positive mutagenic responses were observed at any concentration level or with any tester strain in either the absence or presence of S9 metabolic activation. No test article precipitation was observed. Toxicity was observed as background lawn reduction in the absence of S9 activation starting at 100 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and starting at 1000 μg/plate with tester strain WP2 uvrA. No toxicity was observed in the activated test condition. Only two of the concentration levels scored were non-toxic for tester strains TA98, TA100, TA1535, and TA1537 in the absence of S9 activation. However, the toxicity was observed as background lawn reduction only. All background lawn reductions observed with tester strains TA98 and TA1535 were microscopic. With tester strain TA100 and TA1537, background lawn reduction in the top two dose levels was visible by eye but the third dose level was a microscopic reduction. No reduction in the number of revertant colonies was observed. It was decided that additional testing at lower concentrations was not necessary to make a mutagenicity assessment in this test.

All criteria for a valid study were met with the exception of having less than three non-toxic scorable concentrations as noted above. Under the conditions of this study, H-32217 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9.