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Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of Xanthan lyase, batch PPE55581 was tested in the AMES test and the in vitro micronucleus test.


It was concluded that Xanthan lyase, batch PPE55581 showed no evidence of mutagenic activity in this bacterial system nor did it show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in either of the in vitro test systems described in this section.


 


 

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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.
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 October 2018 - 15 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan locus in the genome of five strains of bacteria.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver.
Test concentrations with justification for top dose:
The OECD Guideline 471 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.

Test 1: 7.9, 23.6, 78.8, 236.4, 788.1, 2364, 7881 μg enzyme concentrate dry matter/mL
Five concentrations of the test item in the final test: (78.8, 236.4, 788.1, 2364 and 7881 enzyme concentrate dry matter/mL).
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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 1x 10^9 cells/mL

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 1 °C for 3 hours (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count and observation of the bacterial background lawn. 0.1 ml aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates in duplicates. The plates were inverted and incubated at 37 ± 2°c for about 72 hours and scored.

Evaluation criteria:
The test substance was considered as positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appertaining solvent control in one or more of the strains, in the presence or absence of S9 mix, if this response is dose related (at least 3 doses) and reproducible. In case of a dose related and reproducible numerical increase, which is below a doubling but at least 50% higher than the solvent control, the result is considered as equivocal and needs further clarification.

Statistics:
Not performed.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but no issues were reported in this study.
- Definition of acceptable cells for analysis: Viability and gene type control

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: Yes

No evidence of toxicity was obtained following exposure to Xanthan lyase, batch PPE55581. No precipitate was observed on any of the plates containing Xanthan lyase, batch PPE55581.

The results with strain TA98 in the presence of S9 mix were obtained from a repeated test. Some increases in revertant colony numbers compared to the concurrent vehicle control counts were observed in strain TA1535 in the absence of S9 mix. The response was flat with no real concentration-response relationship and did not reach 3-fold increase. Hence, the acceptance criteria for a positive response was not fulfilled. The Sponsor indicated that the increases may have arisen from the test item containing various nutrients composing a rich growth medium to the bacterial cultures and therefore stimulating their growth. No evidence of mutagenic activity was seen with any other strains at any concentration of Xanthan lyase, batch PPE55581 in either the presence or absence of S9 mix.

Conclusions:
It was concluded that Xanthan lyase, batch PPE55581 showed no evidence of mutagenic activity in the presence and absence of S9, in this bacterial system under the test conditions employed.
Executive summary:

In this in vitro assessment of the mutagenic potential of Xanthan lyase, batch PPE55581, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to Xanthan lyase, batch PPE55581 diluted in water. Water was also used as a vehicle control.


The mutation tests were performed according to the treat-and-wash method. They were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Two independent mutation tests were performed. Concentrations of Xanthan lyase, batch PPE55581 up to 7881 μg enzyme concentrate dry matter/mL were tested. Other concentrations used were a series of about half-log10 dilutions of the highest concentration.


No signs of toxicity towards the tester strains were observed in either mutation test following exposure to Xanthan lyase, batch PPE55581 in either test. No precipitate was observed on any of the plates containing Xanthan lyase, batch PPE55581. Some increases in revertant colony numbers compared to the concurrent vehicle controls were observed in strain TA1535 in the absence and presence of S9 mix in the first test, and in the absence of S9 mix in the second test. The response was flat with no real concentration response relationship and did not reach 3-fold increase. Hence, the acceptance criteria for a positive response was not fulfilled. The increases may have arisen from the test item containing various nutrients composing a rich growth medium to the bacterial cultures and therefore stimulating their growth. No evidence of mutagenic activity was seen with any other strains at any concentration of Xanthan lyase, batch PPE55581 in either mutation test.


The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory, and within the acceptable ranges defined in Gatehouse et al (1990). At present, there is a limited number of data available for the treat-and-wash method of the Ames test. For this reason, as well as to provide a robust set of current laboratory records, historical data for the standard Ames test are also reported.


It was concluded that Xanthan lyase, batch PPE55581 showed no evidence of mutagenic activity in the presence and absence of S9, in this bacterial system under the test conditions employed.


 


References:


Gatehouse, D.G. et al (1990) Bacterial mutation assays in: Kirkland, D.J. (Ed.). UKEMS


Sub-committee on Guidelines for Mutagenicity Testing. Report. Part I. Basic Mutagenicity


Tests. Cambridge University Press, Cambridge. pp.13-61.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Not classified.