Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Trypsin was tested for genotoxicity potential using Ames and Chromosome Aberration test.

It was concluded that the results of the experiments give no indication of mutagenic activity of SP 387/TL1 in the presence or absence of metabolic activation, up to 5000 µg/mL under the conditions employed in this study.

It is concluded that SP 387/TL1 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 µg/mL, an acceptable maximum concentration for chromosome aberration studies according to current regulatory guidelines, in both the absence and presence of a rat liver metabolic activation system (S-9).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 July 2007 - 28 November 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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 in each test to avoid the possibility that bio-available histidine in the test item might compromise the test.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
The study was performed to assess the effect of the test material SP 387/TL1 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:
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
Test concentrations with justification for top dose:
Six doses 156, 313, 625, 1250, 2500 and 5000 μg substance/mL) were tested.
Vehicle:
- Vehicle(s)/solvent(s) used: DI water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
other: 2-Aminoanthracene, N-Methyl-N´-Nitrosoguanidine
Details on test system and 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 109 cells/mL

DURATION
- Preincubation period: 3 hours
- Exposure duration: Same as preincubation for treat and plate
- Expression time (cells in growth medium): 64 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:
A test substance is considered as positive in this treat and plate assay when it has induced at least a doubling in the mean number of revertants per plate compared to the appropriate solvent control in one or more of the strains, in the presence or absence of S9, if this response is dose related (at least 3 doses) and reproducible. If a numerical increase below a doubling is observed the result is considered as equivocal and need further clarification if the increase is dose related and reproducible and it is not accompanied by significant increases in the viable bacterial count.
Statistics:
N/A
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
Slight cytotoxicity at the highest conc. (+S9)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
Slight cytotoxicity at the highest conc. (+S9)
Vehicle controls valid:
no
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Weak numerical increases were observed in some of the test series. However they were all without any dose relation and coincide with increases in the viable count due to growth stimulating effect of the test substance.
Cytotoxicity was observed at the highest concentration of 5000 μg substance/mL in experiment 1 in strain TA1537 in the presence of S9; and in experiment 2 in strains TA1537 and TA1535 in the presence of S9.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but not an issue in this study
- Definition of acceptable cells for analysis: Viability and gene type control

RANGE-FINDING/SCREENING STUDIES: No

HISTORICAL CONTROL DATA
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes
Conclusions:
It was concluded that the results of the experiments gave no indication of mutagenic activity of SP 387/TL1, batch PPF26813 in the presence or absence of metabolic activation, up to 5000 µg/mL under the conditions employed in this study.
Executive summary:

SP 387/TL 1 (batch PPF26813) was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli W P2uvr A. Crude enzyme preparations, like the present batch of SP387/TL 1, contain the free amino acid histidine, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all Salmonella typhimurium strains were exposed to SP387/TL 1 in liquid culture ("treat and plate assay"). Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg (dry matter) per ml as highest concentration. After incubation the test substance was removed by centrifugation prior to plating.

Usually the content of tryptophan in enzyme preparations is low and insignificant. Therefore the part of the study comprising Escherichia coli was conducted with the strain WP2uvrA using the direct plate incorporation assay. 6 doses of the test substance were applied with 5 mg (dry matter) per plate as the highest dose level followed by successive bi-sections between doses. The study was conducted with and without the metabolic activation system S9 - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix). Two identical and independent experiments were conducted.

A test substance was considered as positive when it had induced at least a doubling in the mean number of revertants per plate compared to the appropriate solvent control in one or more of the strains, in the presence or absence of S9, if this response was dose related and reproducible. If a dose-related numerical increased below a doubling but at least 50% higher than the solvent control was observed then the result was considered as equivocal and would need further clarification.

Cytotoxicity was observed at the highest concentration of 5000 μg substance/mL in experiment 1 in strain TA1537 in the presence of S9; and in experiment 2 in strains TA1537 and TA1535 in the presence of S9. No treatments of any of the Salmonella typhimurium and E. coli strains with SP 387/TL 1, batch PPF26813 either in the presence or absence of S-9 mix, resulted in any increases in revertant numbers that meets these criteria for a positive response.

It was concluded, that the results of the experiments, described in this report, gave no indication of mutagenic activity of SP 387/TL1, batch PPF26813 in the presence or absence of metabolic activation, when tested under the conditions employed in this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Chromosome aberration study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2007- September 12 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests.
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
other: human peripheral blood lymphocytes (HPBL)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL and dilutions hereof.
Experiment 1
(3 hour exposures with 17 hour recovery in the absence and presence of S9):
-S9: Slides evaluated for 2813, 3750, 5000 µg/mL
+S9: Slides evaluated for 2813, 3750, 5000 µg/mL
Experiment 2
(20 hour exposures with 0 hour recovery in the absence of S9):
-S9: Slides evaluated for 3200, 4000, 5000 µg/mL
(3 hour exposures with 17 hour recovery in the presence of S9):
+S9: Slides evaluated for 3200, 4000, 5000 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: Sterile purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and conditions:
METHOD OF APPLICATION: in culture medium
- Cell density at seeding (if applicable): Whole blood cultures

DURATION
- Preincubation period: 48 hours
- Exposure duration: In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17 hour recovery period prior to harvest (3+17). In Experiment 2, treatment in the absence of S-9 was continuous for 20 hours (20+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest (3+17).
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per concentration per experiment.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in mitotic index relative to the vehicle control.
- Determination of endoreplication: Yes
Evaluation criteria:
A test article is considered as positive in this assay if all of the below criteria were met:
1. A proportion of cells with structural aberrations at one or more concentrations exceeded the historical negative control (normal) range in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed at such concentrations
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).

The assay was considered valid if all the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures
2. The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the historical negative control (normal) range
3. At least 160 cells out of an intended 200 were suitable for analysis at each concentration, unless 10 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis
4. The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations.
Statistics:
Fisher's exact test. Probability values of p 0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes (HPBL)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
33% cytotoxicity at 5000 µg/L in Experiment 2 (20+0 h) in the absence of S9
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: N/A
- Water solubility: Yes
- Precipitation: No
- Definition of acceptable cells for analysis: Cytoplasm should be essentially intact. The daughter nuclei were of approximately equal size.

RANGE-FINDING/SCREENING STUDIES: No

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: No
- Indication whether binucleate or mononucleate where appropriate: Yes

HISTORICAL CONTROL DATA
- Positive historical control data: Not stated
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: MI

Conclusions:
It was concluded that SP 387/TL1 batch PPF26813 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 mg/mL, an acceptable maximum concentration for chromosome aberration studies according to current regulatory guidelines, in both the absence and presence of a rat liver metabolic activation system (S-9).
Executive summary:

SP 387/TL1 was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in two independent experiments. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was formulated in sterile water for injection (purified water) and the highest concentration used, 5000 μg/mL, is an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article.

In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17 hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article concentrations for chromosome analysis were selected by evaluating the effect of SP 387/TL1 on mitotic index. Chromosome aberrations were analysed at three concentrations, 2813, 3750, 5000 μg/mL.

In Experiment 2, treatment in the absence of S-9 was continuous for 20 hours (20+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest (3+17). Chromosome aberrations were analysed at three concentrations 3200, 4000, 5000 μg/mL.

Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical vehicle control ranges. 4-Nitroquinoline 1-oxide (NQO) and Cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Treatment of cultures with SP 387/TL1 in the absence and the presence of metabolic activation (S-9) [both experiments] resulted in frequencies of cells with structural chromosome aberrations (excluding gaps), which were similar to those observed in concurrent vehicle control cultures for all concentrations analysed. The aberrant cell frequency of all SP 387/TL1 treated cultures fell within (or very close to) current historical vehicle control (normal) ranges. No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control range were observed in cultures treated with SP 387/TL1 in the absence and presence of S-9 (both experiments).

It was concluded that SP 387/TL1 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 mg/mL, an acceptable maximum concentration for chromosome aberration studies according to current regulatory guidelines, in both the absence and presence of a rat liver metabolic activation system (S-9).

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

Additional information

Justification for classification or non-classification

Not classified.