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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Xanthan Lyase (CAS 113573-69-6, EC name: Xanthan lyase, Enzyme class no 4.2.2.12)
Molecular formula:
n.a.
IUPAC Name:
Active enzyme protein of Xanthan Lyase (CAS 113573-69-6, EC name: Xanthan lyase, Enzyme class no 4.2.2.12)
Constituent 2
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 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:
Carbohydrates as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
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
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPE55581
- Expiration date of the lot/batch: 18 June 2028

Method

Species / strain
Species / strain / cell type:
lymphocytes: cultured human peripheral bloodlymphocytes
Details on mammalian cell type (if applicable):
Add info on the source of cells
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
N/A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The OECD Guideline 487 recommends a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). The test item was tested as 5000 µg total organic solids (TOS)/mL maximum dose, equivalent to 7881 µg enzyme concentrate dry matter/mL, fulfilling the recommendation. 6 lower concentrations were included in the main test.
Vehicle / solvent:
Vehicle for enzyme: Purified water.
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: Cells were placed in HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated fetal calf serum and 0.52% penicillin / streptomycin. The mitogen Phytohaemagglutinin was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.

DURATION
- Preincubation period: Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
- Exposure duration: 3+17h without S-9; 3+17h with S-9; 20+17h without S-9.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Acridine Orange

NUMBER OF REPLICATIONS: A, B. Sets of duplicate cultures were exposed to the test substance.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were centrifuged at 500 g for 5 minutes, the supernatant removed, and the cell pellet re-suspended in the residual supernatant. Pre-cleaned microscope slides were prepared for each culture by aliquoting 50 µL of the cell suspension onto the slides and allowing the slides to air-dry. One slide was prepared from each culture. The slides were then stained using an acridine orange solution at 0.0125 mg/mL in purified water.

NUMBER OF CELLS EVALUATED: 2000 per concentration

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Providing that all of the acceptance criteria have been met, the test item was considered to be
clearly positive if, in any of the experimental conditions examined:

a) At least one of the test concentrations exhibited a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
b) The increase in the frequency of micronucleated cells was dose-related when evaluated with an appropriate trend test.
c) Any of the results were outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).

If all of these criteria were met, the test item was considered able to induce chromosome breaks
and/or gain or loss in the test system.

Providing that all of the acceptance criteria had been met, a negative response would be
claimed if, in all of the experimental conditions examined:

a) None of the test concentrations exhibited a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
b) There was no concentration-related increase when evaluated with an appropriate trend test.
c) All results were inside the distribution of the historical vehicle control data (within the 95% confidence limits).

If all of these criteria were met, the test item was considered unable to induce chromosome
breaks and/or gain or loss in the test system.

DETERMINATION OF CYTOSTASIS
- Method:
Cytostasis = 100-100{(CBPI(T)-1)/(CBPI(C)-1)}, where:
CBPI = (No. mononucleate cells +2 x No. binucleate cells + 3 x No. multinucleate cells)/Total number of cells.
CBPI = cytokinesis-block proliferation index
T = test item treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
To calculate the CBPI at least 500 cells were assessed per culture.
Evaluation criteria:
The concurrent vehicle control must be considered acceptable for addition to the laboratories historical negative control data base (ideally within the 95% confidence limits of current historical control data range). Where concurrent vehicle control data fall outside the 95% confidence limit they may be acceptable for inclusion in the historical control distribution as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ (as assessed using X-bar control charts) and there is evidence of no technical or human failure.

Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent vehicle control.

The criteria for selection of the top dose concentration are consistent with those outlined in the study plan.

Adequate number of cells and concentrations are analyzable.

A minimum of 50% of cells have gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.

Tests that did not fulfill the required criteria were rejected and therefore are not reported.
Statistics:
An arcsine square-root transformation was used to transform the data. Treatment groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using two-tailed t tests.
Statistical significance was declared at the 5% level for all tests.

Results and discussion

Test results
Species / strain:
lymphocytes: from human blood
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: In both the absence and presence of S9 mix, following 3-hour treatment, no marked changes in pH (shifts of greater than 1.0 unit) were observed in post-treatment medium samples any of the test item treated cultures when compared with the concurrent vehicle controls. Increases in osmolality (shifts of greater than 50mOsmol/kg) were observed in post-treatment medium samples at 4000 and 5000 μg TOS/mL in the absence of S9mix, and at 3000, 4000 and 5000 μg TOS/mL in the presence of S9 mix when compared with the concurrent vehicle controls.
- Evaporation from medium: N/A
- Water solubility: Enzymes are water soluble.
- Precipitation: Enzymes are water soluble.
- Definition of acceptable cells for analysis: Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity range finding was conducted.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Not statistically significant
- Indication whether binucleate or mononucleate where appropriate: Yes. Binucleate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: The proportion of micronucleated binucleate cells in the vehicle cultures fell within current 95th percentile of the observed historical vehicle control (normal) ranges.

Applicant's summary and conclusion

Conclusions:
It was concluded that Xanthan lyase, batch PPE55581 did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.
Executive summary:

This study was designed to assess the potential of Xanthan lyase, batch PPE55581 to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro.


The study consisted of a preliminary toxicity test and a main micronucleus test.  Human lymphocytes in whole blood culture, were exposed to Xanthan lyase, batch PPE55581 for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix.  The maximum final concentration to which the cells were exposed was 7881 µg enzyme concentrate dry matter/mL (equivalent to 5000 µg Total Organic Solids (TOS)/mL, dosed at 10% v/v, which is the required dose according to OECD Guideline 487 (2016)).


Vehicle (water; purified by reverse osmosis) and positive control cultures were included in all appropriate test conditions.


In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, Xanthan lyase, batch PPE55581 did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls and there was no evidence of a linear dose-concentration relationship.  The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical control data 95% confidence limits.  These results all fulfilled the criteria for clearly negative results.


The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.


It was concluded that Xanthan lyase, batch PPE55581 did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

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