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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPF 26813
- Expiration date of the lot/batch: Stable at least until 2017 (10 years).
- pH: 6.2
- Stability under test conditions: The prepared formulations were stable for at least 24 hrs when stored at 4ºC or at room temperature in the dark.
- Storage condition of test material: -18°C

Method

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
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.
Controls
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

Results and discussion

Test resultsopen allclose all
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

Applicant's summary and conclusion

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.