Xanthan lyase

Endpoint summary

Key value for chemical safety assessment

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.

These results are further supported by read-across from two in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different enzymes, which have a genotoxicity profile comparable to Xanthan lyase.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Oct. 25, 1993 - Sept. 14, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the similarities between the two enzymes, similar results are expected for Xanthan lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Three types of RPMI 1640 Medium was prepared:
1) RPMI A (consisted of 0 % v/v horse serum, 100 µg/ml Gentamycin, 2.5 µg/ml Fungizone and 0.5 µg/ml Pluronic)
2) RPMI 10 (consisted of 10 % v/v horse serum, 100 µg/ml Gentamycin, 2.5 µg/ml Fungizone and 0.5 µg/ml Pluronic)
3) RPMI 30 (consisted of 20 % v/v horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL (as received) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- The reference chemical Monopropylene glycol (MPG) was also tested because the test chemical formulation of CTGase contains 24 % MPG.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2days
- Selection time (if incubation with a selection agent): At the end of the expression time, the culteres were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable

SELECTION AGENT : 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells using background illumination, expressed as relative survival

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) The mutation frequency at 1 or more doses was significantly greater than that of the negative control.
3) There was a significant dose-relationship as indicated by the linear trend analysis
4) The effects described above were reproducible.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated.

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guideline (Robison et al. (1990), In Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, pp. 102-140). Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. There tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in mutant frequency were observed following treatment with MPG at any dose level as well.

Conclusions:

The amylase CGTase, PPA 4357, IUBMB 3.2.1.1, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (expressed as test material as received) in either the absence or presence of S-9.

Executive summary:

CGTase, PPA 4357 was assayed for its ability to induce mutation at the tk locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a preliminary experiment and cytotoxicity range-finder experiments followed by 2 independent experiments each conducted in the presence and absence of the S-9 mix. The preliminary experiment established that CGTase did not inactivate the enzymes of S-9 mix and therefore it could be tested as supplied.

In the cytotoxicity range-finder experiments 6 doses of CGTase were tested, separated by 2-fold intervals and ranging from 156.25 to 5000 µg/ml. The top dose of CGTase tested yielded 36.1% and 109.6% relative survival in the absence and presence of S-9.

Accordingly, 5 doses of CGTase were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. Four doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top dose plated 5000 µg/ml yielded 91.8% and 90.6% relative survival in the absence and presence of S-9, respectively. In the second experiment 5000 µg/ml CGTase was retained as the top dose but the dose range was modified slightly. The top dose tested in this experiment yielded relative survival values of 95.7% in the absence of S-9 and 116.3% in the presence of S-9.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals. Therefore the study was accepted as valid.

No statistical significant increases in mutant frequency were observed following treatment with CGTase at any dose level either in absence or presence of S-9 in the two experiments.

It was concluded that, under the conditions employed in this study, that the tested amylase CGTase PPA 4357 was not mutagenic in this test system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 1989 - 11 October 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the similarities between the two enzymes, similar results are expected for Xanthan lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fishers medium (10% and 20% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest concentration tested was 5000 µg/mL, tested as supplied (equivalent to 4585 ug enzyme concentrated dry matter/mL).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure is in aqueous solutions.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%)

Evaluation criteria:

A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was evaluated statistically by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

Conclusions:

It was concluded that Lipase had no mutagenic activity in the present test system.

Executive summary:

Lipase was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation (S-9 mix).

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/mL (equivalent to 4585 ug enzyme concentrated dry matter/mL), cells survived this dose of 5000 µg/mL (93% survival) in the absence and 500 µg/mL (11% survival) in the presence of S-9. This, together with the next three lower doses without S-9 and the next five lower doses with S-9, were plated for viability and 6-thioguanine resistance seven days after treatment (with the exception of experiment 1 treatments in the absence of S-9, plated after eight days). In the second experiment a narrower dose range was used to maximise the chance of detecting any lose related effects. The top doses plated in this experiment were 5000 µg/mL in the absence and 500 µg/mL in the presence of S-9, which yielded 104% and 5% survival, respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutation frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore, the study was accepted as valid.

No lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

It was concluded that lipase had no mutagenic activity in this test system.

Reference 3

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.

Reference 4

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)

Justification for classification or non-classification

Not classified.