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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Xanthan lyase was not tested in the Ames Test. However, read-across to two closely related enzymes pectin lyase and pectate lyase was applied. Pectin and pectate lyase were tested for in vitro mutagenic activity in the Ames Test and no mutagenic activity was observed. Due to the close relation between the tested enzyme and xanthan lyase, read-across to xanthan lyase is fully applicable.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vitro, other
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
other information
Justification for type of information:
Two closely related enzymes pectate lyase and pectin lyase have been tested in the Ames Test. Both tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001; Enzymes REACH Consortium, 2009) and the enzyme test material was well characterized. All enzyme classes are hydrophilic and readily biodegradable and in general, non-protease enzymes exhibit the same toxicological properties and although they are potential respiratory sensitizers, they are considered to be of low toxicity, confirmed by toxicity studies performed and published by the industry (summarized in Basketter et al. 2012a and 2012b). The physico-chemical properties of enzymes including logPow are very similar. They are further proteins built up of amino acids and the type, order and number of the amino acids in the polymer differs between enzymes, determining the 3-dimensional structure, the activity and specificity of the individual enzyme type. Industrial production strains typically have a history of safe use for many years in the production of technical and also often food grade enzymes. Because all enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other non-proteolytic enzymes should be fully applicable. Xanthan lyase is concluded not to be mutagenic in the Ames test. References - Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186. - Enzymes REACH Consortium: Safety evaluation of technical enzyme products with regards to the REACH legislation. Document from Manufacturers, Importers and/or Only Representatives of one or more enzymes, who are subject to the registration requirements pursuant to REACH, 2009. http://www.enzymes-reach.org/documents.html - D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012a. J. Immunotox 9(3): 320-6. - Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012b. Reg. Toxicol. Pharmacol, 64/1: 117-123
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 January March 1999 - 19 April 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The two enzymes are very closely related belonging to the same enzyme sub-subclass i.e. 4.2.2. and thus read-across to xanthan lyase can be applied.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
The ‘treat and plate’ treatment method was used.

GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The study describes experiments performed to assess the effect of the test material pectate lyase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535 and TA100); and strain of Escherichia coli WP2uvrApKM101 that can detect substitution at AT to GC or in G-C pair. The test system is a reverse mutation of amino acid dependent bacterial strains.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced Sprague Dawley rats
Test concentrations with justification for top dose:
Six doses 156, 313, 625, 1250, 2500 and 5000 μg/mL were tested.
Vehicle / solvent:
- Vehicle used: DI water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.
- Solvent for positive controls: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene, N-Methyl-N-Nitro-NitrosoGuanidine
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 2 x 10^9 cells/mL

DURATION
- Preincubation period: 3 hrs (liquid culture assay).
- Exposure duration: Same as preincubation for treat and plate
- Incubation time (selective incubation) : 64 hrs

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count
Evaluation criteria:
According to the guideline.
Statistics:
N/A
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA
Not speficied
Conclusions:
Pectate lyase, batch PPE 6345, was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.
Executive summary:

Pectate lyase was tested in two independent experiments. The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test. Six dose levels (156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Pectate lyase was diluted in DI water was added to bacteria growth medium. The bacteria was also treated with the positive controls, respectively. After 3 hr treatment were bacteria washed, plated and incubated for 64 hours. The treatments were performed both in the absence and the presence of metabolic activation system (S-9 mix).

The test item was considered not toxic to the test bacteria, either in the absence or presence of S-9 mix. No increases over 2 -fold in the number of revertant colonies were observed in either experiment.

The results obtained with the solvent and positive controls demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Based on the results obtained in this study, it is concluded that pectate lyase, batch PPE 6345 was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-01-2013 to 09-04-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The two enzymes are very closely related belonging to the same enzyme sub-subclass i.e. 4.2.2. and thus read-across to xanthan lyase can be applied.
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:
S-9 mix from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Preliminary test: Yes
Test 1: 5, 15, 50, 150, 500, 1500, 5000 μg/plate
Test 2: 50, 150, 500, 1500, 5000 μg/plate
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:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
Statistics:
Not 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
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes
Conclusions:
It was concluded that pectin lyase, batch PPJ34366 showed no evidence of mutagenic activity in this bacterial system tested up to a concentration equivalent to 5000 μg/plate in the presence and absence of S9.
Executive summary:

In this in vitro assessment of the mutagenic potential of pectin lyase, batch PPJ34366, 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 pectin lyase, batch PPJ34366 diluted in water. Water was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Both tests included a washing stage for removal of the test substance and any released amino-acids from the treated bacterial cultures, in order to avoid a misleading response caused by growth of pseudorevertant colonies (which may be auxotrophic bacteria, or may be a mixture of prototrophs and auxotrophs) and overgrowth of background lawns.

Pectin lyase, batch PPJ34366 was tested up to a concentration equivalent to 5000 μg/plate (the concentration that would have been applied to the test plate if the original treated culture had been plated without being washed). This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to pectin lyase, batch PPJ34366. No evidence of mutagenic activity was seen at any concentration of pectin lyase, batch PPJ34366 in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising 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.

It was concluded that pectin lyase, batch PPJ34366 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

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

Additional information

Justification for classification or non-classification

Due to the lack of genetic toxicity pectate lyase is not classified.